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Targeting Resistance
Wrestling with cancer can be frustrating. Despite the progress in developing therapies that can effectively control tumor
growth, the devil almost always strikes back with resistance.
Even for the recent excitement in using immunotherapy to
achieve unprecedented success in some cancer patients,
resistance has been seen in clinical settings and is under
active investigation (Restifo et al., 2016).
How do we tackle resistance to cancer therapy? One effective approach is to nail the culpritpinpointing the cell population intrinsically insensitive to the treatment and targeting
their vulnerability. Recent work from Tessa L. Holyoake and
her team successfully applied this strategy on chronic
myeloid leukemia (CML; Abraham et al., 2016). CML is characterized by the aberrant activation of ABL1 tyrosine kinase
due to chromosome translocation, and tyrosine kinase inhibitors (TKIs) have been the standard treatment with clinical
efficacy. However, patients with CML eventually relapse
because the survival of leukemic stem cells (LSCs) does
not rely on the elevated kinase activity and therefore cannot
be eradicated by TKIs. Through integrated analyses, the
team exposed the essential role of p53 and c-MYC on the
CML network and an addictive dependency of LSCs on these
two signaling hubs. They further showed that a combinatory
treatment targeting p53 and c-MYC could effectively kill
LSCs, raising the hope of using this approach for treating
CML patients relapsing from TKIs (Abraham et al., 2016).
Leukemia is not the only type of cancer in which targeting
intrinsic resistance from a specific population is starting to
show promise. Indeed, by developing a mouse model with
knockin reporters, Tannishtha Reya and colleagues were
able to identify high expression of the stem cell determinant
Musashi (Msi) as a marker for populations in pancreatic cancer with strong tumor-initiating capacity and conferring drug
resistance. Inhibiting Msi significantly changed the trajectory
of disease progression and almost doubled the survival time
in mouse models. Moreover, simultaneously inhibiting two of
Jiaying Tan
Cell 166, July 28, 2016 2016 Published by Elsevier Inc. 523
Leading Edge
Conversations
Brain Exploration, Off the Beaten Path
Model organisms, such as rodents, monkeys, or Drosophila, have driven much of recent research
in neuroscience. However, studies in other, more unusual systems have broadened the types
of questions that are being asked and have revealed the diverse ways in which species tackle common problems. Cell editor Mirna Kvajo talked with Nachum Ulanovsky, Gilles Laurent, and Anthony
Leonardo about their research and how studying bats, reptiles, and dragonflies informs big questions in neuroscience. An annotated excerpt of the conversation appears below, and the full conversation is available with the article online.
Anthony Leonardo
Janelia Research Campus
Nachum Ulanovsky
Weizmann Institute of
Science
Gilles Laurent
MPI for Brain Research
we need genomes and all these things. I think . with the advent
of genome editing, it might become a bit less of an issue.
AL: I started working on an unusual non-genetic system in an
era right when genetic systems were really exploding, and it
was clear that it was tactically not the wisest decision in terms
of certain aspects. And the thing that always sort of struck me is
that the genetic access to these sorts of weirdo systems is only
going to get easier over time, whereas the computations the
animals do and the behaviors they do are fixed. So its not that
mice and flies are going to evolve new behaviors suddenly that
youre going to be able to study in them. So there is a real
reason and a utility in saying, OK, this organism is solving this
computation, and this is a good place to study it, and were
going to work on it at the level of tools we have now, and
gradually more tools will become available and well gain
deeper levels of understanding it. As opposed to forcing that
problem into a genetic system where its very hard to study and
you make progress very slowly, even with the elegance of the
tools there.
GL: You could also turn this around by saying that many of
the tools that we use now actually come from the study of
unusual systems like bioluminescence in jellyfishyou get GFP
and the channelrhodopsins and CRISPR/Cas9. That doesnt
come from directed research at the beginning; its really
curiosity driven. If we lose this, we lose a lot of these
advantages.
AL: Yeah, I think on that same token, it is interesting to notice
that a lot of the problems being studied at a deep mechanistic
level in our genetic systems are problems that were described
at the level of algorithms and principles in other systems
things that we used to study in Hoverflies and locusts and all
these sort of exotic creatures that are being tapped. They
provided essentially the foundation on which these more
mechanistic studies can be built in other systems. And that
really arises from the ubiquity of evolution and the fact that
these principles do transcend the system, and almost by
definition you should be able to look at these things in different
places, and the breadth and depth can combine effectively.
I want to add yet another component, another advantage of
maintaining this diversity and studying non-standard species:
the natural behaviors. I mean, you cannot study a laboratory rat
or laboratory mouse in the wild. You can study a wild rat or wild
mice, which in some of their behaviors are quite different than
the ones in the laboratory. Whereas these non-standard
organisms, they are literally wild animalsliterally, we capture
them from the wild. You can study them also outdoors, so
weve been studying the bats, GPS tracking them outdoors,
looking at their navigation, etc. I think this really opens your
thinking to asking different questions. You see a behavior
outdoors and start asking, How is that implemented? And
you can eventually bring it to a laboratory setting and do a
controlled experiment, but even being able to study the animal
out in the wild is something that you typically cannot do. Or
even if you can, its not done by most people on standard
laboratory animals.
Leading Edge
Voices
Big Questions in Evolution
Evolution of Cell Types and Tissues
Detlev Arendt
Nicole King
Sean B. Carroll
528 Cell 166, July 28, 2016 2016 Published by Elsevier Inc.
Patricia Wittkopp
Eugene Koonin
Leonid Kruglyak
University of Michigan
UCLA, HHMI
Leading Edge
Previews
In Praise of Descriptive Science:
A Breath of Fresh AIRE
Mark M. Davis1,*
1Howard
Hughes Medical Institute, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford,
CA 94305, USA
*Correspondence: mmdavis@stanford.edu
http://dx.doi.org/10.1016/j.cell.2016.07.018
Meyer et al. find that subjects lacking the AIRE gene, critical for self-tolerance in T lymphocytes,
show a broad range of autoantibody specificities, which can have extremely high affinities. The
data also suggest that some of these autoantibodies can, surprisingly, prevent some types of autoimmunity, particularly type I diabetes.
In molecular biology, there has been a
persistent bias against the value of
descriptive biology. This might go back
to a remark attributed to Ernest Rutherford that There are two kinds of science;
physics and stamp collecting. Of course,
the real power in any scientific area
comes with a precise knowledge of
mechanisms, but it shouldnt be forgotten
that careful observation and discovering
new phenomena is the starting point of
every field. A case in point is the paper
in this issue of Cell by Meyer et al.
(2016), who take advantage of new technologies that are allowing us to get precise data about the human immune system to discover some remarkable and
unexpected properties of human beings
with a particular immune deficiency.
They start, innocently enough, with a
straightforward enquiry into the nature of
the autoantibodies in subjects that are
deficient in what is known as the AIRE
gene, which was originally identified in
APS1an autoimmune syndrome characterized by autoantibodies, impaired
endocrine function, and chronic Candida
infections (Nagamine et al., 1997;
Finnish-German APECED Consortium,
1997). Later work in mice has shown
that Aire has a specific role in stimulating
the expression of tissue-specific genes
in the thymus that wouldnt normally be
expressed in that organ and that this
helps ensure T cell tolerance to self-antigens (Mathis and Benoist, 2009). There
is also evidence that Aire has a role in
ensuring T cell tolerance in peripheral immune organs such as the spleen and
lymph nodes (Gardner et al., 2013). Tolerance is induced at least in some cases by
clonal deletion of self-specific T cells (Anderson et al., 2005) but also might take the
form of inhibiting activation (Davis, 2015).
In some mouse strains, Aire deficiency
leads to severe autoimmunity and early
death, but in other strains and in human
beings, the effects are more subtle. In
this study, the authors gathered specimens and data on 81 patients. Then they
analyzed the autoantibodies in their
serum for specificity. Remarkably, they
found that each patient expresses on
average approximately 100 different
specificities, such that, all together, they
were able to identify over 3,700 antibody
specificities, showing that there was an
almost random pattern of targets. However, there were some specificities that
were shared, particularly anti-cytokine
antibodies to cytokines in the type I interferon group. Even more remarkable is
that, when they characterized some of
these autoantibodies with respect to
their affinity, they found that many were
astonishing high, with one having a KD of
10 14M and others in the picomolar range
(10 12M), much higher than the nanomolar affinities that one gets with a standard
immunization. They further note that patients with this syndrome seem resistant
to many autoimmune diseases (multiple
sclerosis, lupus, and others) but a fraction
do develop type I diabetes. While there is
a growing literature correlating anti-cytokine antibodies with a susceptibility to
particular infectious diseases (Kisand
et al., 2010), they postulated that such autoantibodies might, in some cases, have a
protective effect. In particular, a-interferons have been implicated in type I diabetes in mice, and so they looked to see
530 Cell 166, July 28, 2016 2016 Published by Elsevier Inc.
human mutations can uncover new phenomena worthy of further study. This not
only opens up new vistas regarding our
understanding of immune function and
dysfunction but also shows how work
on the human immune system can not
only inform translational work but add to
our understanding of basic principles as
well.
REFERENCES
Anderson, M.S., Venanzi, E.S., Chen, Z., Berzins,
S.P., Benoist, C., and Mathis, D. (2005). Immunity
23, 227239.
Davis, M.M. (2015). Immunity 43, 833835.
Finnish-German APECED Consortium. (1997). Nat.
Genet. 17, 399403.
Gardner, J.M., Metzger, T.C., McMahon, E.J., AuYeung, B.B., Krawisz, A.K., Lu, W., Price, J.D., Johannes, K.P., Satpathy, A.T., Murphy, K.M., et al.
(2013). Immunity 39, 560572.
Kisand, K., Be Wolff, A.S., Podkrajsek, K.T.,
Tserel, L., Link, M., Kisand, K.V., Ersvaer, E., Perheentupa, J., Erichsen, M.M., Bratanic, N., et al.
(2010). J. Exp. Med. 207, 299308.
Mathis, D., and Benoist, C. (2009). Annu. Rev. Immunol. 27, 287312.
Meyer, S., Woodward, M., Hertel, C., Vlaicu, P.,
Haque, Y., Karner, J., Macagno, A., Onuoha,
S.C., Fishman, D., Peterson, H., et al. (2016). Cell
166, this issue, 582595.
Nagamine, K., Peterson, P., Scott, H.S., Kudoh, J.,
Minoshima, S., Heino, M., Krohn, K.J., Lalioti,
M.D., Mullis, P.E., Antonarakis, S.E., et al. (1997).
Nat. Genet. 17, 393398.
Sabouri, Z., Schofield, P., Horikawa, K., Spierings,
E., Kipling, D., Randall, K.L., Langley, D., Roome,
B., Vazquez-Lombardi, R., Rouet, R., et al.
(2014). Proc. Natl. Acad. Sci. USA 111, E2567
E2575.
Leading Edge
Previews
Broadening Horizons:
New Antibodies Against Influenza
Katherine J.L. Jackson1 and Scott D. Boyd1,*
1Department of Pathology, Stanford University, CA 94305, USA
*Correspondence: sboyd1@stanford.edu
http://dx.doi.org/10.1016/j.cell.2016.07.023
Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation.
Two studies now report engineered or naturally occurring human antibodies targeting the influenza
hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al.,
2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that
modified vaccine regimens could provide broad protection.
To paraphrase Jane Austen, it is a truth
universally acknowledged that an individual in possession of a good broadly
neutralizing antibody response to a virus
must be in want of characterization. The
human immune response to influenza
has been no exception. The tools of
monoclonal antibody (mAb) characterization from single B cells, and tracking of the
clonal evolution of B cell populations by
high-throughput antibody sequencing,
are providing an increasingly high-resolution map of human immunity to a range of
pathogens and vaccines (Andrews et al.,
2015; Liao et al., 2013). Influenza is a significant global health challenge with millions of infections and up to half a million
deaths globally each year, despite efforts
to increase vaccination in at-risk populations. Influenza virus is a challenging
target for antibodies because of antigenic
drift and shift, genetic processes that produce ongoing and occasionally abrupt
antigenic changes. Antibodies elicited
by seasonal influenza vaccination tend
to target the rapidly mutating globular
head of hemagglutinin (HA), facilitating
viral escape from responses raised to
other viral strains (Krammer and Palese,
2013). Broadly neutralizing antibodies
(bnAbs) directed against the structurally
conserved HA stem could provide more
enduring protection, and examples of
these have been isolated (Krammer and
Palese, 2013). These antibodies have revealed some stereotyped features, such
as IGHV1-69 usage in multiple subjects
(Corti et al., 2010). These responses in vivo
in humans, however, seem to arise from
low-frequency clones, are present at
lower titers than head-specific antibodies,
REFERENCES
Andrews, S.F., Huang, Y., Kaur, K., Popova, L.I.,
Ho, I.Y., Pauli, N.T., Henry Dunand, C.J., Taylor,
W.M., Lim, S., Huang, M., et al. (2015). Sci. Transl.
Med. 7, 316ra192.
The pragmatic goal of better vaccine efficacy against divergent viral strains could
be served equally well by eliciting rare antibodies with extreme breadth or a handful
of antibody lineages with complementary
partial breadth. The success of each scenario could depend on the titers reached
and the durability of the response. Evaluating the feasibility of these goals will
likely require testing vaccine regimens using heterologous antigens that are more
divergent than those in current vaccine
formulations and assessing antigenic
combinations, the ordering of antigen
administration, and the effects of adjuvants, while analyzing the kinds of antibodies that are stimulated in each case.
Of course, very potent bnAbs are potential passive immunoprotective or thera-
Leading Edge
Previews
Baby Nuclear Pores Grow Up Faster All the Time
C. Patrick Lusk1,*
1Yale School of Medicine, New Haven, CT 06510, USA
*Correspondence: patrick.lusk@yale.edu
http://dx.doi.org/10.1016/j.cell.2016.07.011
Annulate lamellae (AL) are stacked ER-derived membranes containing nuclear pore complex-like
structures whose fate and function have remained a mystery. During the short interphase of early
embryonic cells, AL are rapidly delivered into the nuclear envelope through fenestrations, highlighting the remarkable dynamics of the nuclear envelope.
The confinement of the genome within the
nucleus suggests that it, like most organelles, is a physically distinct cellular entity.
However, the two nuclear membranes
that comprise the nuclear envelope (NE)
are made up of one lipid bilayer that is
contiguous with the endoplasmic reticulum (ER) (Figure 1). The morphological
and biochemical identity of the NE is
classically defined by the presence of nuclear pore complexes (NPCs), which are
enormous 100 MD transport channels
composed of nucleoporin (nup) proteins.
Importantly, NPCs do not disrupt the continuity of the lipid bilayer between the NE
and ER but effectively seal NE pores
by controlling the passage of soluble
and membrane-bound macromolecules
into and out of the nucleus. Interestingly,
the morphological distinction between
NE and ER is blurred in some cell types,
as extra-nuclear NPC-like structures
occur in stacked ER cisternae (Cordes
et al., 1996). These annulate lamellae
(AL) were considered repositories of
excess nups, but this function was not
formally established. Moreover, it is challenging to contemplate how (or whether)
AL could be incorporated into the NE
without breaking the NE seal that would
also impose a topological barrier to such
an event. In this issue of Cell, Hampoelz
et al. (2016) show how rapidly dividing
cells in the Drosophila embryo overcome
this barrier by visualizing an elegant
mechanism of AL incorporation into the
NE. In addition to providing a solution to
the long-standing question of the function and fate of AL, they also introduce
a mechanism of membrane remodeling
that is challenging our conventional view
of a static interphase NE.
A common feature of early embryonic
divisions is their rapidity, necessitating
uniquely dynamic NE-ER system. Consistent with this idea, as the embryonic cells
progressively immobilize NPCs by the
increased expression of intranuclear scaffolds like the lamins and lamin-binding
proteins, they lose the capacity to incorporate AL into the NE. Thus, AL incorporation may be halted as cell fate and NE
composition become cemented. Alternatively, AL incorporation might be slowed
because of a requirement to remodel a
rigid intranuclear lamin scaffold, a likely
necessity for NE dynamics in highly differentiated cells with more established nuclear architecture (King and Lusk, 2016).
A model in which the AL incorporate
into the NE makes the prediction that the
NE and AL together comprise a compartment that is distinct from the rest of the
ER. Remarkably, and consistent with this
idea, the permeability barrier of the NE
to macromolecules remains intact despite
the presence of the NE fenestrations, suggesting that the AL effectively seal the
NE like a lid that would be part of a
larger, yet-to-be defined NE-AL system
(Figure 1). A major challenge for the future
is to understand how membranes competent for AL formation either remain connected with the NE during mitotic NE
breakdown or establish connections by
generating holes in a sealed NE. A likely
possibility for the former scenario is that,
during NE breakdown, some membranes
(perhaps those that associate with the
mitotic spindle) retain a biochemical or
morphological signature of the NE that
establishes competence for NE reformation and NPC assembly. The future
identification of this molecular signature
might represent the minimal fundamental
component that triggers NPC assembly
and provides the ultimate differentiator
of the NE and ER.
tion of NPC assembly upon AL incorporation into the NE (Figure 1). This NPC
maturation must rely on positional
cues that could reflect binding to chromatin (Franz et al., 2007; Rasala et al.,
2006) or to components of the nuclear
basket known to be required for NPC assembly but missing from AL (Vollmer
et al., 2015). Thus, one exciting possibility is that the maturation mechanism
reflects conformational changes to the
NPC scaffold that expose otherwise
hidden anchor points for the remaining
nups. One wonders whether this process
might be reversed during NPC disassembly or dynamically controlled to alter
the transport properties of the NPC in
response to environmental (or other) inputs. In either case, understanding the
molecular basis behind these putative
conformational changes will likely be a
watershed in our understanding of NPC
assembly and function.
REFERENCES
Cordes, V.C., Reidenbach, S., and Franke, W.W.
(1996). Cell Tissue Res. 284, 177191.
Leading Edge
Previews
A Biomarker Harvest
from One Thousand Cancer Cell Lines
Yu-Han Huang1 and Christopher R. Vakoc1,*
1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
*Correspondence: vakoc@cshl.edu
http://dx.doi.org/10.1016/j.cell.2016.07.010
Identifying molecular biomarkers that predict cancer drug efficacy is crucial for the advancement of
precision medicine. In this issue of Cell, Iorio et al. nominate hundreds of potential genetic and
epigenetic biomarkers through high-throughput drug screening in 1,000 molecularly annotated
cancer cell lines.
Developing personalized therapies that
exploit the unique molecular abnormalities in a patients tumor is a central objective of modern cancer research. Genome
sequencing initiatives, such as The
Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium
(ICGC), have defined the complex genetic
landscapes of the most common human
malignancies; however, only a small
percentage of cancer mutations is
considered to be actionable with existing
therapies (Garraway and Lander, 2013;
Stratton et al., 2009). While new targets
and drugs are clearly needed, a major
obstacle in implementing precision therapies is our incomplete understanding of
the relationship between tumor genotype
and drug sensitivity. To address this
issue, many investigators have turned to
large-scale chemical screens in genetically annotated human cancer cell lines
as a means of nominating predictive biomarkers (Figure 1). While cancer cell lines
in culture are imperfect models of human
tumors, they tend to remain addicted to
the oncogenes that initiated tumor formation and hence are well-validated tools for
studying oncogene-targeted therapies
(Sharma et al., 2010). In this issue, Iorio
et al. present one of the largest attempts
to date at mining predictive correlates of
drug sensitivity using a panel of 1,000
annotated cancer cell lines treated with
265 compounds (Iorio. et al., 2016).
The strategy taken by the authors is the
following: (1) perform a deep genetic and
epigenetic analysis of each cell line,
focusing on the features that match the
recurrent alterations found in human tumors, (2) measure the sensitivity of each
cell line to 265 different compounds/
ACKNOWLEDGMENTS
C.R.V. is supported by the Leukemia and Lymphoma Society, the Burroughs-Wellcome Fund,
the Pershing Square Sohn Cancer Research Alliance, the Starr Cancer Consortium, and the NIH/
NCI grant RO1 CA174793.
REFERENCES
Barretina, J., Caponigro, G., Stransky, N., Venkatesan, K., Margolin, A.A., Kim, S., Wilson, C.J., Lehar, J., Kryukov, G.V., Sonkin, D., et al. (2012). Nature 483, 603607.
Chabner, B.A. (2016). J. Natl. Cancer Inst.
108, 108.
Garraway, L.A., and Lander, E.S. (2013). Cell 153,
1737.
Haibe-Kains, B., El-Hachem, N., Birkbak, N.J., Jin,
A.C., Beck, A.H., Aerts, H.J., and Quackenbush, J.
(2013). Nature 504, 389393.
Iorio, F., Knijnenburg, T.A., Vis, D.J., Bignell, G.R.,
Menden, M.P., Schubert, M., Aben, N., Goncalves,
E., Barthorpe, S., Lightfoot, H., et al. (2016). Cell
166, this issue, 740754.
Kaelin, W.G., Jr. (2005). Nat. Rev. Cancer 5,
689698.
Luo, J., Solimini, N.L., and Elledge, S.J. (2009). Cell
136, 823837.
Seashore-Ludlow, B., Rees, M.G., Cheah, J.H.,
Cokol, M., Price, E.V., Coletti, M.E., Jones, V.,
Bodycombe, N.E., Soule, C.K., Gould, J., et al.
(2015). Cancer Discov. 5, 12101223.
Sharma, S.V., Haber, D.A., and Settleman, J.
(2010). Nat. Rev. Cancer 10, 241253.
Stratton, M.R., Campbell, P.J., and Futreal, P.A.
(2009). Nature 458, 719724.
Leading Edge
Review
The Genetics of Transcription Factor
DNA Binding Variation
Bart Deplancke,1,* Daniel Alpern,1 and Vincent Gardeux1
1Laboratory
of Systems Biology and Genetics, Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne and Swiss Institute of
Bioinformatics, 1015 Lausanne, Switzerland
*Correspondence: bart.deplancke@epfl.ch
http://dx.doi.org/10.1016/j.cell.2016.07.012
Most complex trait-associated variants are located in non-coding regulatory regions of the
genome, where they have been shown to disrupt transcription factor (TF)-DNA binding motifs. Variable TF-DNA interactions are therefore increasingly considered as key drivers of phenotypic variation. However, recent genome-wide studies revealed that the majority of variable TF-DNA binding
events are not driven by sequence alterations in the motif of the studied TF. This observation implies
that the molecular mechanisms underlying TF-DNA binding variation and, by extrapolation, interindividual phenotypic variation are more complex than originally anticipated. Here, we summarize
the findings that led to this important paradigm shift and review proposed mechanisms for local,
proximal, or distal genetic variation-driven variable TF-DNA binding. In addition, we discuss the
biomedical implications of these findings for our ability to dissect the molecular role(s) of noncoding genetic variants in complex traits, including disease susceptibility.
Introduction
Analysis of genomic variation in humans (Auton et al., 2015) as
well as in model species such as the mouse (Keane et al.,
2011; Yalcin et al., 2011) and fruit fly (Huang et al., 2014; Massouras et al., 2012) is providing unprecedented opportunities to
understand the genetic basis of complex traits, including disease
susceptibility. An important insight that emerged from genomewide association studies (GWAS) is that the vast majority of
significantly associated genetic variants is located in non-coding
regions and may thus impact gene regulation. For example, of
465 unique trait/disease-associated single nucleotide polymorphisms (SNPs) derived from 151 GWAS studies, only 12% are
located in protein-coding regions, while 40% fall within introns
and another 40% in intergenic regions (Hindorff et al., 2009). In
addition, genome-wide profiling of accessible chromatin regions
using DNase I hypersensitivity (DHS) mapping revealed that
almost 60% of non-coding GWAS SNPs and other variants are
located within DHS sites, with another 20% being in complete
linkage disequilibrium (LD) with variants that lie in a proximate
DHS site (Maurano et al., 2012). Since DHS sites reflect the occupancy of DNA binding proteins such as transcription factors
(TFs), these data indicate that GWAS loci may alter the binding
of TFs and, as such, induce variation in gene expression and
ultimately in complex organismal phenotypes. In this Review,
we summarize the findings that led to this increasingly accepted
notion of the importance of variation in TF-DNA binding in mediating phenotypic diversity. In addition, we strive to clarify why, for
the majority of studied traits or diseases, establishing a mechanistic link between regulatory and phenotypic variation is still
very challenging.
For this purpose, we explore the molecular mechanisms mediating TF-DNA binding variation and address a major question in
538 Cell 166, July 28, 2016 2016 Elsevier Inc.
Table 1. Examples Linking Variable TF-DNA Binding to Phenotypic Variation Arranged by Date of Characterization
Causal Variant Position
Relative to TSS
Phenotype
Affected Gene
HBG
175 bp
Haemophilia B Leyden
F9
20 bp; 10 bp;
6 bp
Affected Binding
Site
TFBS
Outcome
GATA1; TAL1
Gain
HNF4a; C/EBPa;
OC1/OC2
Loss
Reference(s)
Haemophilia B Brandenburg
F9
26 bp
AR
Loss
Delta-thalassemia
HBD
77 bp
GATA1
Loss
DARC
46 bp
GATA1
Loss
LPL
39 bp
OCT1
Loss
Bernard-Soulier syndrome
GP1BB
133 bp
GATA1
Loss
Osteoporosis
COLlAl
Sp1
Gain
HNF1A
58 bp
HNF4A
Loss
Asthma
IL10
509 bp
YY1
Gain
PKLR
72 bp
GATA1
Loss
UROS
70 bp;
GATA1; CP2
Loss
Psoriasis
SLC9A3R1
237 bp
RUNX1
Loss
FASLG
844 bp
CEBPB
Loss
Esophageal cancer
COX-2
1195 bp
c-MYB
Gain
TCOF1
346 bp
YY1
Loss
Alpha-thalassemia
HBA
13 bp
GATA1
Gain
Holoprosencephaly
SHH
460 kb
SIX3
Loss
Various cancers
TERT
187 bp
ETS2
Loss
IRF6
14 kb
AP2
Loss
SOX9
1.44 Mb
MSX1
Loss
Prostate cancer
MYC
200 kb
FOXA1
Gain
Colorectal cancer
MYC
300 kb
TCF7L2
Gain
ZPBP2;
GSDMB;
ORMDL3
CTCF
Loss
Myocardial infarction
SORT1
44 kb
CEBPA
Loss
Beta-thalassemia
HBB
71 bp
GATA1
Loss
F7
60 bp
HNF4A
Loss
Osteoarthritis
GDF5
41 bp
YY1
Loss
Breast cancer
CCND1
127 kb;
ELK4; GATA3
Loss; Gain
TERT
+2bp;
ETS2
Gain
KITLG
+20 kb
Hirschsprung disease
SOX10
Insulin resistance
PPARG2
ARAP1
Melanoma
SDHD
25 bp;
Pancreatic agenesis
PTF1A
25 kb
TAL1
7.5 kb
IRX3; IRX5
0.5 Mb;
Colorectal cancer
FASLG
1377 bp;
+2 kb
90 bp
76 kb
66 bp;
88 bp
P53
Loss
30 kb
AP2; SOX10
Loss
6 kb
PRRX1
Loss
PAX6/PAX4
Loss
Loss
FOXA2, PDX1
Loss
MYB
Gain
ARID5B
Loss
SP1; STAT1
Loss
+418 bp
7 bp;
4 bp
1.2 Mb
670 bp
TFs may still be able to bind to this core motif, albeit with much
lower affinity. This may, in part, explain the observed discrepancy between in vivo DNA occupancy levels and in-vitro-derived
DNA binding affinities (Biggin, 2011), since these in vivo binding
events may reflect binding by interacting TF pairs and not individual TFs. It is therefore clear that a large portion of motifs
remain to be characterized, emphasizing the need for new technologies or efforts to close this gap.
Imputing DNA Binding Variation
It has often proven difficult to infer whether a specific polymorphism will significantly change TF-DNA binding and act as a
regulatory variant, even if the PWM model of the TF is available.
This complication stems from difficulties in capturing the
DNA binding complexity of a TF in a robust binding model either
to confidently detect a genuine binding site within a given
sequence or to accurately infer the impact of a variant on detected motifs.
The Accuracy of Binding Models and Robustness of Motif
Detection. The majority of motif detection methodologies rely
on PWM representation since PWMs perform relatively well
with respect to capturing the overall binding affinity. This is
because PWMs can be modeled as a numerical matrix, which
enables the scoring of a given sequence according to its similarity to a motif (Figure 1). Nevertheless, it is important to acknowledge that this model also has several limitations, which may
impede the discovery of the correct binding patterns. For
example, PWM models assume that the nucleotide binding energies are independent (Stormo and Zhao, 2010), which proved
not to be generally valid (Bulyk et al., 2002; Jolma et al., 2013;
Maerkl and Quake, 2009; Nutiu et al., 2011), and are also suboptimal to represent the binding of TF dimers, since many of these
542 Cell 166, July 28, 2016
bind to two sequences that are separated by a spacer with variable length. These caveats have spurred the development of
different models for representing TF motifs, such as hidden Markov models (HMMs) (Gelfond et al., 2009; Zhao et al., 2005) and
more advanced machine learning models, stimulated by the
increasing availability of multiple layers of genomic, transcriptomic, and epigenomic information. Among these are support
vector machine (SVM) or neural network (NN) approaches that
are trained on datasets containing both known regulatory and
random sequences, with the goal of recognizing and scoring
new putative regulatory sequences (Gao and Ruan, 2015)
(Figure 1 and Table S1). Such representations have many advantages over conventional models because they are highly flexible.
In addition, they are not limited to the DNA sequence recognized
by the TF and can incorporate additional features that are also
important to model TF-DNA binding. These features include
the 3D structural conformation of DNA and its steric characteristics (Levo and Segal, 2014; Rohs et al., 2009; Zhou et al., 2015),
the chemical properties used to model TF amino acid-nucleotide
contacts at the atomic level (Bauer et al., 2010; MaienscheinCline et al., 2012), protein concentration (Djordjevic et al.,
2003; Wang and Batmanov, 2015) that allows for a more accurate estimation of DNA occupancy and thus intrinsic DNA binding affinity (Biggin, 2011; Simicevic et al., 2013), and, finally,
the nucleotide composition of motif-neighboring sequences.
Indeed, recent work revealed that the sequence environment
of a genuine binding site tends to be distinct from that of unbound sequences. In particular, it was shown to exhibit specific
sequence features such as high GC content (White et al., 2013)
or a higher similarity to the core motif (Dror et al., 2015) that
may guide TFs to their cognate binding sites. These findings
events had a SNP directly located in the NFKB motif and induced
a binding difference that was consistent with its perceived
impact on motif quality (i.e., reduced binding was linked to a
SNP that lowered the PWM binding score and vice versa)
(Kasowski et al., 2010). One of the possible reasons that were
listed (next to LD or putative epigenomic variation) involved
trans-effects. However, ChIP-seq analyses of >20 TFs revealed
extensive, allele-specific DNA binding (in a constant trans
environment), effectively refuting this hypothesis (Reddy et al.,
2012). Subsequent studies in human LCLs and in cells or
tissues derived from distinct mouse strains observed a similar
pattern (Heinz et al., 2013; Kilpinen et al., 2013; Soccio et al.,
2015; Stefflova et al., 2013), collectively emphasizing the importance of cis-regulatory variation. Importantly, only a minority
of differential allelic occupancy events involved nucleotide
changes in the respective motifs (Reddy et al., 2012). However,
this does not mean that variation in the motifs of other TFs
should also be dispensed as a possible molecular mechanism for these observationsquite the contrary, in fact, as we
will clarify in greater detail in the next paragraphs (see also
Figure 2).
If a particular genetic variant does not affect the motif of the
studied TF, what then causes the respective TF to exhibit differential DNA binding? It appears that an important fraction (at least
7.5% according to our own estimate [Kilpinen et al., 2013]) of
variable TF-DNA binding events can be explained by alterations
of proximal motifs (Reddy et al., 2012). Thus, at some genomic
sites, TFs appear to be dependent on the proximal presence of
other TFs to bind to DNA. Qualitative motif analysis combined
with prior knowledge about the biological process in which the
focal TF is operational lends credibility to this notion. For
example, in mouse white adipose tissue, PPARg binding sites
that vary between strains and do not harbor an altered PPARg
motif were analyzed for enriched, polymorphic motifs. The topscoring motifs corresponded to the TFs CEBPa and glucocorticoid receptor (Soccio et al., 2015) that exhibit extensive
co- localization with PPARg in mature white fat cells (Siersbk
et al., 2014). Similarly, differential PU.1 binding correlated with
544 Cell 166, July 28, 2016
alterations in the motifs for the TFs CEBP and AP-1, which
modulate macrophage activity (Heinz et al., 2013). However,
this correlation appears to differ according to macrophage subtype. Indeed, a follow-up study in mouse microglia revealed that
other TF motifs correlate better with variable PU.1 DNA binding,
emphasizing the importance of cellular context in determining
this type of TF interactions (Gosselin et al., 2014). Together,
these studies strongly support the notion of pervasive DNA binding whose occurrence is dependent on the presence of other
TFs. Since it is well appreciated that regulatory regions tend to
harbor binding sites for multiple TFs, this notion may not be
entirely surprising. Nevertheless, it is worthwhile in the current
context of genetic variation to briefly revisit this mode of DNA
binding, which is interchangeably called cooperative or collaborative DNA binding (Gosselin et al., 2014; Mirny, 2010; Slattery
et al., 2014; Waszak et al., 2015). We would thereby like to argue
that, for the sake of discussion and molecular understanding, it
might be valuable to differentiate between these two terms
(Figure 2).
Local, Cooperative TF-DNA Binding
In the context of protein-DNA interactions, cooperativity was
initially used in describing the assembly of E. coli lambda repressors on DNA (Ptashne et al., 1980). Binding of a lambda
dimer on a first operator site facilitates binding of another
lambda dimer on the second operator site, given that physical
interactions between the first and second dimer increase the
affinity of the latter for DNA, which explains why cooperative
DNA binding is evoked to define this process. Consequently,
the term cooperativity may be especially suited for DNA binding
processes that involve TFs whose physical interactions at the
protein level may increase the affinity of the entire complex to
specific sites in the genome. For example, binding of the winged
HTH DNA binding domain-containing TF IRF4 is cooperatively
enhanced by the TF PU.1 (Escalante et al., 2002). This is
because binding of the two TFs contorts the DNA in a peculiar
S shape, placing the TFs in an optimal position for electrostatic
and hydrophobic interactions and thus stabilizing the entire
complex (Escalante et al., 2002). Consequently, individual
stacked or overlapping molecular phenotypes that did not correlate with other neighboring molecular phenotypes. Thus, a totem
VCM represents local chromatin state variation (Figure 3A). The
remaining multi-VCMs are more interesting since, while a
minority, they typically cover two or more distinct regulatory elementshence the term multiand capture the majority of all
detected molecular phenotypes (Figure 3A). The origin of a
multi- VCM is less intuitive than that of a totem-VCM. Its structure suggests, however, a higher-order chromatin organization
that is reminiscent of the modular genomic structure that has
been uncovered in the form of topologically associating domains
(TADs) (Dixon et al., 2012; Nora et al., 2012).
These TADs constitute distinct, three-dimensional genomic
structures in which sequences are more likely to interact with
one another than with those located outside the respective
TAD. However, VCMs and TADs constitute different molecular
entities because VCMs tend to be embedded within TADs and
thus tend to be smaller (Waszak et al., 2015) (Figure 3B). In addition, TADs are relatively stable across cell types and during
development and are even conserved across species (Dixon
et al., 2012; Vietri Rudan et al., 2015), whereas VCMs are by definition variable. As such, multi-VCMs correspond conceptually
better to sub-TADs, which are more fine-grained (sub-Mb),
genomic topologies that have been shown to be dynamic across
cellular differentiation (Dixon et al., 2015; Phillips-Cremins et al.,
2013) and to even differ between individual cells (Giorgetti et al.,
2014). In addition, sub-TADs have been suggested to define cisregulatory networks (Berlivet et al., 2013), with their internal
conformational dynamics being directly related to embedded
transcriptional activity (Giorgetti et al., 2014; Tang et al., 2015).
In parallel, the vast majority of gene-associated multi-VCMs exhibited a molecular activity state that significantly correlated with
the transcriptional activity of the included gene(s) (Waszak et al.,
2015) (Figure 3B). Moreover, the more regulatory elements
encompassed in a VCM, the more likely it was to associate
with variable gene expression. Together, the conceptual similarities between sub-TADs and multi-VCMs suggest that the latter
also reflect fine-grained configurations of interacting regulatory
elements with one or a few target genes whose collective, molecular activity is highly coordinated. As such, VCMs may
provide substantial insights into the structural and thus modular
organization of the chromatin landscape, including TF-DNA
interactions.
Which mechanisms lie at the origin of multi-VCMs? Since the
long-range molecular coordination that typifies multi-VCMs has
been observed at the allelic level (Kasowski et al., 2013; Kilpinen
et al., 2013; McVicker et al., 2013) and since recent chromatin
interaction analysis by paired-end tag sequencing (ChIA-PET)
data has also provided evidence for allele-specific chromatin topologies (Tang et al., 2015), it is reasonable to assume that the
observed molecular variation is largely driven by genetic factors.
Moreover, most of the molecular variation within each VCM
could be captured by a single, quantitative phenotype (Waszak
et al., 2015), which suggests that the activity state of a VCM
can be attributed to relatively few but strong causal variants.
QTL mapping using the activity state of each VCM as input
yielded vcmQTLs that were highly enriched in TF-occupied regions (Waszak et al., 2015) (Figure 3A). Together with previous
tfQTL and cQTLs, but not with eQTLs. There are several complementary hypotheses that could explain the existence of such
island VCMs, including (1) futile regulatory activity without
transcriptional consequences (Cusanovich et al., 2014; Farnham, 2009; Wasserman and Sandelin, 2004); (2) regulatory
redundancy, which prevents a gene-specific regulatory network
from collapsing even if one node or edge is impacted (Pai et al.,
2015), consistent with the shadow enhancer concept (Hong
et al., 2008); (3) regulatory regions that are not transcriptionally
operational, at least in the studied condition/cellular environment, which implies that the activity of these regions is tissue
specific. Indeed, if, in a hypothetical study, a complex trait-associated regulatory variant would be linked to an island VCM, it
might indicate that an incorrect system or context is being
studied, as its disconnection with gene expression is unlikely
to yield a cellular or organismal phenotype. This reasoning is
consistent with the observation across several studies that
GWAS variants tend to be most enriched for eQTLs in tissues
that are relevant to the phenotype (Emilsson et al., 2008; Nica
et al., 2010; Torres et al., 2014). However, in most cases, the
causal variants are obviously unknown a priori. To identify
them, it may prove valuable to, similar to eQTLs, map VCMs in
as many distinct cell types/tissues as possible. The resulting
set of VCMs may then provide guidance to both variant identification and characterization. Indeed, the most interesting candidates among the set of associated GWAS variants would be
those that impact not only on the chromatin topology (e.g.,
vcmQTLs) and state of the respective locus (e.g., cQTLs or
tfQTLs), but also on expression of the embedded gene. Once
identified, it should be relatively straightforward to detangle the
underlying molecular mechanisms since the coordinated, molecular phenotypes that make up the focal VCM should provide
clear insights into the flow of regulatory information, i.e., from
causal nucleotide over gene to ultimately cellular or organismal
phenotype.
DNA in machine learning approaches. In addition, it is increasingly appreciated that the chromatin context needs to be accounted for when searching for causal, regulatory variants and
that, in general, the use of cell types or systems that are most
relevant for the studied trait or disease will yield the best results.
It is also important to recognize that only a small fraction of all
variable TF-DNA binding events is actually driven by variation
within the motif of the studied TF. Thus, similar to gene expression, TF-DNA binding is a complex molecular trait by itself, which
has profound implications for our understanding of how regulatory variation arises.
Well-established concepts in the gene regulation field provide
an intuitive molecular foundation for local or proximal variantdriven DNA binding variation. Specifically, the former involves
cooperative DNA binding that is mediated by direct, physical
interactions between TFs, while the latter appears to be driven
by collaborative DNA binding that is likely reflective of sequenceor chromatin-context conditioned TF interdependencies to
displace nucleosomes and open chromatin. However, the mechanisms that underlie distal variant-driven DNA binding changes
are much less well understood (Figure 2). The identification of
3C-, ChIA-PET-, or VCM-based chromatin entities that link
structural information to transcriptional function is important in
this regard since they offer a molecular rationale to explain these
prevalent, long-range DNA binding dependencies. Sustained
efforts will therefore be required to unravel the modular structure
of the variable (epi)genome across a wide range of cells or
tissues. Thus, although many challenges remain, exciting progress is being made in elucidating the genetic basis of TF-DNA
binding variation that will undoubtedly improve our ability to
achieve a nucleotide-level understanding of the molecular mechanisms underlying many complex traits, including disease
susceptibility.
Conclusions
The fundamental discovery that most complex trait-associated
variants are located in non-coding, putatively regulatory regions
of the genome has focused the spotlight on TF-DNA interactions
as important mediators of phenotypic variation. Yet, to date,
relatively few examples are available in which a clear mechanistic relationship between TF-DNA binding variation and phenotypic variation was established (Table 1). To clarify why this is
such a challenging task, we focused in this Review on elucidating
how the impact of genetic variation on TF-DNA binding can be
assessed and why, contrary to expectations, this is itself already
inherently complex. There are several current limitations that will
have to be addressed to improve our ability to identify and interpret regulatory variation, including the need for new experimental or computational approaches that will enable us to
expand the TF motif catalog, to better predict genuine TF binding
sites, and to evaluate how motif variation affects TF-DNA binding. Promising research avenues in this regard include the development of new technologies to characterize monomeric and
higher complex TF-DNA binding properties and the incorporation of additional DNA binding features such as the sequence
environment and the conformational and chemical nature of
Supplemental Information includes one table and is available with this article
online at http://dx.doi.org/10.1016/j.cell.2016.07.012.
SUPPLEMENTAL INFORMATION
ACKNOWLEDGMENTS
We thank Richard Benton (University of Lausanne), Sebastian Waszak (EMBL),
Alina Isakova, Antonio Meireles-Filho, Petra Schwalie, and other members of
the Deplancke Laboratory, as well as the anonymous reviewers for useful comments on the manuscript. We also would like to acknowledge scientific discussions with all members of the Effect of sequence variation on chromatin
structure and transcription Sinergia Consortium (i.e., the Reymond and Hernandez Laboratories [UNIL] and the Dermitzakis Laboratory [University of
Geneva]). This work was supported by the Swiss National Science Foundation
grant CRSI33_130326, by SystemsX.ch (AgingX, 51RTP0_151019), and by
institutional support from the Swiss Federal Institute of Technology in Lausanne (EPFL).
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Leading Edge
Review
Mitochondria and Cancer
Sejal Vyas,1 Elma Zaganjor,1 and Marcia C. Haigis1,*
1Department
of Cell Biology, Ludwig Center at Harvard, Harvard Medical School, Boston, MA 02115, USA
*Correspondence: marcia_haigis@hms.harvard.edu
http://dx.doi.org/10.1016/j.cell.2016.07.002
Mitochondria are bioenergetic, biosynthetic, and signaling organelles that are integral in stress
sensing to allow for cellular adaptation to the environment. Therefore, it is not surprising that mitochondria are important mediators of tumorigenesis, as this process requires flexibility to adapt to
cellular and environmental alterations in addition to cancer treatments. Multiple aspects of mitochondrial biology beyond bioenergetics support transformation, including mitochondrial biogenesis and turnover, fission and fusion dynamics, cell death susceptibility, oxidative stress regulation,
metabolism, and signaling. Thus, understanding mechanisms of mitochondrial function during
tumorigenesis will be critical for the next generation of cancer therapeutics.
Introduction
Historical Perspective
Louis Pasteur identified the importance of oxygen consumption
in 1861, finding that yeast divided more in the presence of oxygen and that oxygen inhibited fermentation, an observation
known as the Pasteur effect. The discovery of mitochondria
in the 1890s, described cytologically by both Richard Altmann
and Carl Benda, began to shed light on this observation, and in
1913, the biochemist Otto Warburg linked cellular respiration
to grana derived from guinea pig liver extracts (Ernster and
Schatz, 1981). Warburg stated that the granules functioned to
enhance the activity of iron-containing enzymes and involved a
transfer to oxygen (Ernster and Schatz, 1981). In the following
decades, many scientists elucidated the machinery that drives
mitochondrial respiration, including tricarboxylic acid (TCA)
cycle and fatty acid b-oxidation enzymes in the mitochondrial
matrix that generate electron donors to fuel respiration and electron transport chain (ETC) complexes and ATP synthase in the inner mitochondrial membrane (IMM) that carry out oxidative
phosphorylation (Ernster and Schatz, 1981). This biochemical
understanding of mitochondrial oxidative phosphorylation gave
mechanistic insight into the Pasteur effect, which could be reconstituted by adding purified, respiring liver mitochondria to
glycolytic tumor supernatants and observing inhibited fermentation (Aisenberg et al., 1957). The ability of mitochondria to inhibit
a glycolytic system suggested an active and direct role for mitochondria in regulating oxidative versus glycolytic metabolism
(Aisenberg et al., 1957).
Warburgs seminal discovery that cancer cells undergo aerobic glycolysis, which refers to the fermentation of glucose to
lactate in the presence of oxygen as opposed to the complete
oxidation of glucose to fuel mitochondrial respiration, brought
attention to the role of mitochondria in tumorigenesis (Warburg,
1956). While the Warburg effect is an undisputed feature of
many (but not all) cancer cells, Warburgs reasoning that it
stemmed from damaged mitochondrial respiration caused immediate controversy (Weinhouse, 1956). We now understand
that while damaged mitochondria drive the Warburg effect in
some cases, many cancer cells that display Warburg metabolism possess intact mitochondrial respiration, with some cancer subtypes actually depending on mitochondrial respiration.
Decades of studies on mitochondrial respiration in cancer have
set the framework for a new frontier focused on additional functions of mitochondria in cancer, which have identified pleiotropic
roles of mitochondria in tumorigenesis.
A major function of mitochondria is ATP production, hence its
nickname powerhouse of the cell. However, mitochondria
perform many roles beyond energy production, including the
generation of reactive oxygen species (ROS), redox molecules
and metabolites, regulation of cell signaling and cell death, and
biosynthetic metabolism. These multifaceted functions of mitochondria in normal physiology make them important cellular
stress sensors, and allow for cellular adaptation to the environment. Mitochondria similarly impart considerable flexibility for tumor cell growth and survival in otherwise harsh environments,
such as during nutrient depletion, hypoxia, and cancer treatments, and are therefore key players in tumorigenesis.
There is no simple canon for the role of mitochondria in cancer
development. Instead, mitochondrial functions in cancer vary
depending upon genetic, environmental, and tissue-of-origin
differences between tumors. It is clear that the biology of mitochondria in cancer is central to our understanding of cancer
biology, as many classical cancer hallmarks result in altered
mitochondrial function. This review will summarize functions of
mitochondria biology that contribute to tumorigenesis, which
include mitochondrial biogenesis and turnover, fission and
fusion dynamics, cell death, oxidative stress, metabolism and
bioenergetics, signaling, and mtDNA (Figures 1 and 2).
Mitochondrial Biogenesis and Turnover
Mitochondrial mass is dictated by two opposing pathways,
biogenesis and turnover, and has emerged as both a positive
and negative regulator of tumorigenesis. The role of mitochondrial biogenesis in cancer is regulated by many factors, including
metabolic state, tumor heterogeneity, tissue type, microenvironment, and tumor stage. Additionally, mitophagy, the selective
Cell 166, July 28, 2016 2016 Elsevier Inc. 555
Figure 3. Effects of Classical Oncogenic and Tumor Suppressive Pathways on Mitochondrial Biology
Key mechanisms of mitochondrial regulation by c-MYC, K-RAS, PI3K, and p53 signaling pathways. Through transcriptional regulation, c-Myc induces mitochondrial biogenesis and metabolism in addition to its stimulation of cell-cycle progression and glycolysis. c-Myc promotes mitochondrial fusion and respiration,
which can result in increased ROS production and oxidative signaling. Hyperactive PI3K signaling through either PI3K mutation or loss/mutation of the PTEN
tumor suppressor results in mTOR activation, which is additionally regulated by nutrient availability, to regulate cell growth. mTOR promotes mitochondrial
biogenesis both transcriptionally and translationally. Low nutrient conditions that result in a high AMP/ATP ratio activate AMPK, which opposes the mTOR
pathway. During chronic nutrient deprivation, AMPK can also promote mitochondrial biogenesis to allow for metabolic flexibility. Loss of p53 promotes survival
not only via transcriptional regulation of cell death programs, but also through direct interactions with Bcl-2 proteins at the mitochondria. p53 can also induce
mitochondrial respiration to promote tumorigenesis by allowing for metabolic flexibility. Oncogenic K-Ras mutations result in a coordinated program of mitochondrial regulation, reprogramming mitochondrial metabolism through multiple mechanisms as well as promoting mitochondrial fission and mitophagy.
Mitophagy
Clearance of damaged mitochondria via mitophagy is critical for
cellular fitness since dysfunctional mitochondria can impair ETC
function and increase oxidative stress. A major trigger for
mitophagy is via the PTEN-induced putative kinase 1 (PINK1)/
Parkin pathway. This pathway is activated upon mitochondrial
membrane depolarization, a signal of mitochondrial dysfunction
that results from multiple causes including lack of reducing
equivalents, hypoxia, and impaired electron transport. An alternate pathway for mitophagy induction is through the HIF-1a
target genes Bcl-2 and adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L/NIX), which inhibit mitochondrial respiration during hypoxic conditions that could result
in excessive ROS.
558 Cell 166, July 28, 2016
Is mitophagy beneficial or harmful to cancers? Similar to autophagy, which is shown to be both pro- and anti-tumorgenic
based on context, the function of mitophagy in transformation
likely depends on tumor stage (Mancias and Kimmelman,
2016). Mitophagy-deficient Parkin null mice develop spontaneous hepatic tumors, and Parkin loss increases tumorigenesis
in multiple cancer models (Matsuda et al., 2015). Additionally,
BNIP3 and NIX are identified as tumor suppressors in multiple
cancer models (Chourasia et al., 2015). Thus, in certain stages
of tumorigenesis, decreased mitophagy may allow for a permissive threshold of dysfunctional mitochondria to persist, generating increased tumor-promoting ROS or other tumorigenic
mitochondrial signals. In contrast, established tumors may
require mitophagy for stress adaptation and survival. Supporting
Cell Death
A hallmark of cancers is their ability to evade cell death, a phenomenon tightly linked to mitochondria. The pro-apoptotic
Bcl-2 family members Bax and Bak are recruited to the OMM
and oligomerize to mediate mitochondrial outer membrane permeabilization (MOMP), resulting in pore formation and cytochrome c release from mitochondria into the cytosol to activate
caspases, the executors of programmed cell death. During
normal physiology, anti-apoptotic family members such as
Bcl-2 and Bcl-XL bind and inhibit Bax/Bak. Tumor cells escape
apoptosis by downregulating pro-apoptotic Bcl-2 genes and/or
upregulating anti-apoptotic Bcl-2 genes, achieved through multiple mechanisms reviewed elsewhere (Lopez and Tait, 2015).
The balance of pro- and anti-apoptotic proteins affects a cancer
cells susceptibility to apoptotic stimuli and may predict how a
tumor will respond to chemotherapy (Sarosiek et al., 2013).
Mitochondrial shape also dictates apoptotic susceptibility, as
Drp1 loss delays cytochrome c release and apoptotic induction,
although follow-up work indicated that fission was not required
for Bax/Bak-mediated apoptosis (Martinou and Youle, 2011).
Instead, a GTPase-independent function of Drp1 in membrane
remodeling and hemifusion results in Bax oligomerization and
subsequent MOMP, indicating that Drp1 can promote apoptosis
independent of fission (Martinou and Youle, 2011). The
importance of mitochondrial shape in apoptosis is further demonstrated by Mfn-1-loss induced mitochondrial hyperfragmentation, causing resistance to apoptotic stimuli due to the loss of
Bax interaction with mitochondrial membranes. In this study,
Drp1 inhibition rescued sensitivity to apoptotic stimuli by
restoring a balanced mitochondrial network (Renault et al.,
2015). Additionally, Mfn1 is a target of the MEK/ERK signaling
pathwayphosphorylated Mfn1 inhibits mitochondria fusion
and interacts with Bak to stimulate its oligomerization and
subsequent MOMP (Pyakurel et al., 2015). Therefore, while fission
and fusion do not necessarily regulate apoptosis per se, a balance
of these activities appears to generate a mitochondrial shape that
supports interactions with pro-apoptotic Bcl2 proteins.
Oxidative Stress
ROS, in the form of superoxide and hydroxyl free radicals, and
hydrogen peroxide, are produced from physiological metabolic
reactions. Mitochondria are major contributors to cellular ROS
and have multiple antioxidant pathways to neutralize ROS
including superoxide dismutase (SOD2), glutathione, thioredoxin, and peroxiredoxins. The early observation that cancer
cells have high ROS levels led to an overly simple hypothesis
that inhibiting ROS could be a successful therapeutic strategy.
However, a more complex picture is emerging, in which ROS
stimulates signaling and proliferation, and the concomitant
upregulation of antioxidant pathways prevents ROS-mediated
cytotoxicity and may even enhance tumor survival (Shadel and
Horvath, 2015; Sullivan and Chandel, 2014).
Multiple physiological reactions, including electron transport
by the ETC and NAD(P)H oxidases result in ROS production,
and these are often exacerbated during tumorigenesis by
oncogenic signaling, ETC mutations, and hypoxic microenvironments. High levels of ROS contribute to the oxidation of macromolecules, such as lipids, proteins, and DNA, and can contribute
Cell 166, July 28, 2016 559
An important signaling pathway in hypoxic tumor microenvironments is mediated by HIF-1a, which upregulates glycolytic
metabolism in low oxygen conditions and inhibits mitochondrial
respiration (Mucaj et al., 2012). Mitochondrial-derived ROS also
regulate the HIF-1a pathway via inhibition of prolyl hydroxylases (PHDs), negative regulators of HIF signaling. SIRT3, a
mitochondrial deacetylase, is an important regulator of this
pathway by maintaining redox homeostasis via deacetylation
and activation of mitochondrial SOD2 and IDH2 and indirectly
through transcriptional upregulation of antioxidant pathways
(Bause and Haigis, 2013). SIRT3-dependent reduction in mitochondrial ROS results in HIF-1a degradation, limiting glycolysis
and the Warburg effect in tumors (Bell et al., 2011; Finley et al.,
2011).
In addition to the pleiotropic effects of oncogenic K-Ras
signaling on proliferation, apoptosis, and metabolism, oncogenic K-Ras results in a coordinated program of mitochondrial
regulation that supports transformation (Pylayeva-Gupta et al.,
2011). Multiple K-Ras-dependent mechanisms can downregulate mitochondrial respiration including upregulation of mitochondrial fission (Kashatus et al., 2015; Serasinghe et al.,
2015), transcriptional downregulation of complex I (Wang et al.,
2015), and ERK-phosphorylation-dependent mitochondrial
translocation of phosphoglycerate kinase I (PGK1) (Li et al.,
2016). Oncogenic K-Ras also promotes upregulation of mitophagy to preserve mitochondrial function under starvation conditions. Autophagy inhibition in cancers with active K-Ras results
in a decline in mitochondrial respiration, TCA metabolite, and
energy levels during starvation; thus, this pathway may be
important for tumor cell survival in nutrient-depleted microenvironments (Guo et al., 2011).
The PI3K/Akt signaling pathway stimulates cell growth and is
often activated in cancer either through oncogenic mutations
in signaling kinases or loss/mutation of the PTEN tumor suppressor, a key phosphatase that shuts off this pathway (Papa et al.,
2014). Although PI3K signaling induces cell growth and upregulates glycolysis, metabolic adaptation via a switch to mitochondrial oxidative phosphorylation can mediate resistance to PI3K
inhibitors, undermining the effectiveness of PI3K-specific targeted therapy (Ghosh et al., 2015). A major downstream effector
of active PI3K/Akt signaling is mTOR, which participates in
mTORC1 and mTORC2 signaling complexes to couple nutrient
and growth-factor sensing to cellular growth through regulation
of translation, anabolic metabolism, and autophagy (Dibble
and Cantley, 2015). In addition to regulating mitochondrial
biogenesis, mTORC1 stimulates multiple mitochondrial metabolic pathways. The transcriptional repression of SIRT4 downstream of mTORC1 activity results in GDH activation to
upregulate glutaminolysis (Csibi et al., 2013). mTORC1 also induces the mitochondrial folate pathway to promote de novo
purine synthesis via activation of the transcription factor ATF4
to result in upregulation of MTHFD2 expression (Ben-Sahra
et al., 2016).
The AMP-regulated kinase (AMPK) signaling network is activated during low energy conditions, directly inhibiting multiple
targets including mTORC1 to restore energy homeostasis.
AMPK is a critical downstream target of the liver kinase B1
(LKB1) tumor suppressor, which is mutated in the inherited
562 Cell 166, July 28, 2016
mitochondrial-derived citrate is used by histone acetyl transferases (HATs), and oncogenic signaling pathways modify histone
acetylation patterns in a ACLY-dependent manner (Lee et al.,
2014; Wellen et al., 2009). In addition to chromatin modification,
acetyl-CoA generated from mitochondrial-derived citrate is used
for the acetylation of many cytosolic and mitochondrial proteins
to modulate protein activity. Thus, mitochondrial-derived metabolites can effect signaling pathways, nuclear transcription, and
chromatin modification.
In addition to signaling molecules, readouts of mitochondrial
integrity including Dcm and MOMP also function as important
signals, enabling the cell to respond to unhealthy/dysfunctional
mitochondria. Since the membrane potential generated by
healthy mitochondria is required for protein import into the mitochondrial matrix and intermembrane space via the TIM22 and
TIM23 translocator complexes, loss of membrane potential impairs import. If the defect in protein import is severe, the cell
can initiate mitophagy to clear these unhealthy mitochondria
as discussed above. Additionally, ATP generation by the ETC
is an important signaling output with diminished ETC activity
increasing the AMP/ATP ratio to activate AMPK signaling. ETC
dysfunction can also result in decreased NAD+ levels, a co-substrate for both the sirtuin and poly(ADP-ribose) protein families,
which have many functions in tumorigenesis (German and Haigis, 2015; Vyas and Chang, 2014). Finally, ROS regulates cytosolic signaling networks to promote tumorigenesis (as discussed
above).
Mitochondrial Oncometabolites
Dominant mutations in mitochondrial enzymes led to the exciting
identification of mitochondrial-derived signaling molecules,
termed oncometabolites. Mutant versions of cytoplasmic and
mitochondrial IDH isoforms, found in a striking 20% of acute
myeloid leukemias and 70% of glioblastomas, reduce a-KG to
generate the oncometabolite (R)-2-hydroxyglutarate ([R]-2-HG)
(Dang et al., 2009; Ward et al., 2010). In addition, loss of function
of TCA cycle enzymes succinate dehydrogenase (SDH) and
fumarate hydratase (FH), underlying the inherited cancer predispositions hereditary paraganglioma syndrome and hereditary
leiomyomatosis and renal-cell cancer syndrome, respectively,
result in the accumulation of metabolic intermediates succinate
and fumarate, which function as oncometabolites when in
excess.
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their structural similarity to a-KG, allowing them to act as
competitive inhibitors of a-KG-dependent enzymes including
the TET and JHDM families of chromatin-modifying enzymes
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near gene promoters, which results in gene silencing (Nowicki
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marks on H3K9 and H3K27 are observed in IDH1 and IDH2
mutant gliomas due to JHDM inhibition (Lu et al., 2012). Therefore, through the production of oncometabolites, mitochondria
exert strong influence on chromatin structure to promote
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Article
Authors
Eunhee Choi, Xiangli Zhang, Chao Xing,
Hongtao Yu
Correspondence
hongtao.yu@utsouthwestern.edu
In Brief
Mitotic checkpoint proteins that prevent
chromosomes from incorrectly
segregating have another unexpected
job: they regulate insulin signaling. This
role draws a link between chromosome
stability and nutrient metabolism.
Highlights
d
Article
Mitotic Checkpoint Regulators Control
Insulin Signaling and Metabolic Homeostasis
Eunhee Choi,1 Xiangli Zhang,2 Chao Xing,2 and Hongtao Yu1,*
1Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road,
Dallas, TX 75390, USA
2Bioinformatics Core, Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center,
6001 Forest Park Road, Dallas, TX 75390, USA
*Correspondence: hongtao.yu@utsouthwestern.edu
http://dx.doi.org/10.1016/j.cell.2016.05.074
SUMMARY
delete p31comet in the whole body (Figures S1AS1C). Homozygous p31comet knockout (p31/) mice were born in the expected
Mendelian ratio. Heterozygous p31+/ mice did not show
568 Cell 166, 567581, July 28, 2016
Figure 2. p31comet Ablation Causes Insulin Resistance, whereas Bub1b Insufficiency Enhances Insulin Sensitivity
(A) Glycogen synthase (GS) activity of WT, liver-p31/, and liver-Insr/ hepatocytes treated without () or with (+) insulin (Ins). Mean SD (n = 4 independent
experiments).
(B) Immunoblots of whole-liver lysates of WT, liver-p31/, and liver-Insr/ mice treated without () or with (+) insulin (Ins). Each lane contains lysate from an
individual mouse. The relative band intensities are quantified and shown below.
(C and D) Insulin signaling in primary WT and liver-p31/ hepatocytes treated with 10 nM insulin for the indicated times (C) or increasing concentrations of insulin
for 5 min (D). Cell lysates were blotted with the indicated antibodies.
(E) Segmentation plots of euploid (WT) and aneuploid hepatocytes from liver-p31/ and Bub1bH/H mice.
(FH) Fed blood glucose levels (F), glucose tolerance test (G), and insulin tolerance test (H) of WT and liver-p31/ mice injected with AAV-GFP or AAV-p31. WT
(AAV-GFP), n = 10; liver-p31/ (AAV-GFP), n = 8; liver-p31/ (AAV-p31), n = 7; mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus liver-p31/ (AAV-GFP);
py < 0.05, yyp < 0.01 versus WT (AAV-GFP).
(I) Segmentation plots of aneuploid cells from liver-p31/ mice injected with AAV-GFP or AAV-p31.
(JL) Fed blood glucose levels (J), glucose tolerance test (K), and insulin tolerance test (L) of WT and Bub1bH/H mice. For GTT and ITT, at least 12 mice in each
group were analyzed. Mean SEM. *p < 0.05, **p < 0.01.
See also Figure S3.
level of functional IR at the PM, due to the premature internalization of IR prior to insulin stimulation.
MAD2 Directly Binds to a Canonical MIM Motif in IR
We wondered whether p31comet function in regulating IR internalization involved MAD2. MAD2 had been reported as an IR-binding protein in yeast two-hybrid and proteomic screens (Hutchins
et al., 2010; ONeill et al., 1997). MAD2 binds to MAD1 and
CDC20 through the MAD2-interacting motif (MIM) with the
consensus of (K/R)ccXcX3-4P (c, a hydrophobic residue;
X, any residue; Figure 4A). The C-terminal cytoplasmic tail of IR
contained a putative MIM (Figure 4A). A peptide containing this
motif (IRMIM-WT) efficiently bound to purified recombinant
MAD2 WT and a monomeric mutant R133A (Figure 4B), but did
not interact with MAD2 DC, a truncation mutant that could not
form C-MAD2. A mutant IR peptide (IRMIM-4A) did not interact
with MAD2. Immunoprecipitation with a C-MAD2-specific antibody (Fava et al., 2011) confirmed that IR-bound MAD2 formed
C-MAD2 (Figure 4C). IRMIM-WT bound to C-MAD2R133A with a
dissociation constant of 380 nM (Figure 4D). Thus, IR contains
a functional MIM.
MAD2 WT, but not DC, interacted with the cytoplasmic
domain of IRb (IRb-C) (Figure 4E), indicating that the MIM is
not masked in the intact cytoplasmic domain of IR and is available for MAD2 binding. MAD2 binding did not affect the kinase
activity of IR, and vice versa. Furthermore, IR-WT-MYC interacted with endogenous MAD2 in 293FT cells, whereas IR-4AMYC did not (Figure 4F). Depletion of p31comet enhanced the
IR-MAD2 interaction, suggesting that p31comet might promote
IR-C-MAD2 disassembly. Endogenous MAD2 and IR interacted
with each other in HepG2 cells (Figure 4G) and in whole-liver lysates of WT and liver-p31/ mice (Figure 4H). Unlike in 293FT
cells depleted of p31comet, in liver lysates of liver-p31/ mice
we did not observe a significant increase of the IR-MAD2 interaction, suggesting that p31comet might not actively promote IRMAD2 disassembly in hepatocytes.
A previous report showed that insulin-stimulated IR activation
reduced the IR-MAD2 interaction in IR-overexpressing CHO
cells (ONeill et al., 1997). In contrast, we found that the IRMAD2 interaction in vitro and in human cells was not regulated
by insulin or IR autophosphorylation (Figures 4E4G). Thus, IR
binds to MAD2 through a canonical MIM in vitro and in vivo.
This interaction is constitutive and is not regulated by insulin.
p31comet Regulates IR Endocytosis by
Counteracting MAD2
We generated HepG2 cells stably expressing IR-4A-GFP and
examined the subcellular localization of this MAD2-binding-defi-
(B) HepG2 cells stably expressing IR-WT-GFP were transfected with the indicated siRNAs, serum starved, treated without or with 80 mM dynasore (Dyn), and
stained with indicated antibodies. Scale bars, 10 mm.
(C) Quantification of the ratios of PM and IC IR-GFP signals of cells in (B) (mean SD; *p < 0.0001).
(D) Liver sections of WT, liver-p31/, and liver-Insr/ mice injected with PBS or insulin (+Ins) were stained with anti-IR (red) and anti-RAB7 (green) antibodies and
DAPI (blue). Scale bars, 10 mm. Asterisks indicate sinusoids.
(E) Liver sections of WT and liver-p31/ mice injected with AAV-GFP or AAV-p31 were stained with anti-IR (red) antibodies and DAPI (blue). Scale bars, 10 mm.
(F) Representative images of endocytosis assays with Cy3-insulin and Alexa 568-transferrin in WT and liver-p31/ hepatocytes at 20 min. The insulin and
transferrin signals are shown in red. The DAPI signals are shown in blue. Scale bar, 10 mm.
(G and H) Endocytosis of insulin (G) and transferrin (H) in WT and liver-p31/ hepatocytes. The intensities of internalized fluorescent signals at the indicated times
were quantified (mean SD; n = 3 independent experiments with >70 cells analyzed at each time point).
(E) GST pull-down assays with recombinant GST-IRb-C and MAD2. Input and beads-bound proteins were blotted with the indicated antibodies. The relative
intensities of pY and MAD2 (mean SEM; n = 3 independent experiments) are shown below.
(F) 293FT cells were co-transfected with IR-WT-MYC or IR-4A-MYC constructs and the indicated siRNAs, serum starved, and treated without () or with (+)
100 nM insulin (Ins) for 20 min. The total cell lysate (TCL) and anti-MYC immunoprecipitate (IP) were blotted with the indicated antibodies.
(G) HepG2 cells were transfected with siLuc or siMAD2, serum starved, and treated without () or with (+) 100 nM insulin (Ins) for 20 min. TCL, anti-MAD2 IP, and
IgG IP were blotted with the indicated antibodies.
(H) Total liver lysates from WT, liver-p31/ and liver-Insr/ mice, and anti-MAD2 and IgG IP from these lysates were blotted with the indicated antibodies.
Figure 5. p31comet Suppresses Spontaneous IR Endocytosis through Counteracting the IR-MAD2 Interaction
(A) HepG2 cells stably expressing IR-WT-GFP or IR-4A-GFP were transfected with siLuc or si-p31, serum starved, and stained with anti-GFP (IR; green) antibody
and DAPI (blue). Scale bars, 10 mm.
(B) Quantification of the ratios of PM and IC IR-GFP signal intensities in (A) (mean SD; ****p < 0.0001).
(C) WT and p31/ MEFs were infected with IR-WT-GFP or IR-4A-GFP retroviruses, serum starved, treated without or with dynasore (Dyn), and stained with antiGFP (IR; green) antibody and DAPI (blue). Scale bar, 10 mm.
(D) Quantification of the ratios of PM and IC IR-GFP signal intensities in (C) (mean SD; ****p < 0.0001).
(E) IR-GFP-expressing HepG2 cells were co-transfected with the indicated siRNAs and plasmids and stained with anti-GFP (IR; green) and anti-MYC (p31comet;
red) antibodies and DAPI (blue). Scale bar, 10 mm.
(F) Quantification of the ratios of PM and IC IR-GFP signal intensities in (E) (mean SD; ****p < 0.0001).
(G) Liver sections of WT and liver-p31/ mice injected with Ad-GFP, Ad-IR-WT, or Ad-IR-4A were stained with anti-IR (red) antibody and DAPI (blue). Scale
bar, 10 mm.
Figure 6. p31comet Prevents MAD2- and BUBR1-Dependent IR Endocytosis through Blocking AP2 Recruitment
(A) IR-GFP-expressing HepG2 cells were transfected with the indicated siRNAs, treated without or with 100 nM insulin (Ins) for 5 min, and stained with DAPI (blue)
and anti-GFP (IR; green) and anti-RAB7 (red) antibodies. Scale bars, 10 mm.
whether changes in insulin signaling contribute to the aging phenotypes of mice with altered BUBR1 expression.
Liver-specific ablation of p31comet in mice produces metabolic
disorders reminiscent of type 2 diabetes, including insulin resistance. The underlying mechanisms of insulin resistance in type 2
diabetes are complex and not fully understood (Samuel and
Shulman, 2012). Our findings presented herein implicate premature IR internalization prior to insulin binding as a potential mechanism underlying insulin resistance. It will be interesting to
examine IR levels or localization in liver biopsies of human
type 2 diabetes patients.
Our results establish the role of mitotic checkpoint regulators
in insulin signaling and metabolic homeostasis. Our study provides a striking example of how an entire branch of key regulators in one cellular process can be repurposed to control
another. By virtue of its ability to link signaling pathways originating from kinetochores and the plasma membrane, the
p31comet-MAD2-BUBR1 module may offer a potential conduit
for extracellular hormones to regulate chromosome segregation
and karyotypes.
EXPERIMENTAL PROCEDURES
Generation and Phenotypic Analyses of p31/ and Liver-p31/ Mice
All animal experiments were performed in accordance with institutional guidelines and with approval from the Institutional Animal Care and Use Committee
of UT Southwestern Medical Center.
The strategy of targeting p31comet (Mad2l1bp) is described in Figure S1. The
p31F/F mice were crossed with transgenic mice expressing Cre recombinase
under the Actin promoter to generate the p31/ mice and were mated with
transgenic mice expressing Cre recombinase under the Albumin promoter
to generate liver-p31/ mice. See the Supplemental Experimental Procedures
for information about the mouse crosses and husbandry and generation of
liver-p31/;Bub1b/ mice.
Tissue histology and immunohistochemistry were performed by an oncampus core facility. Hepatic glycogen content was measured with the
Glycogen Assay Kit (Sigma). Glucose and insulin tolerance tests, metabolic
profiling, glycogen synthase activity assay, and in vivo insulin signaling assay
were performed with established protocols. Prism was used for the generation
of all curves and graphs and for statistical analyses. See the Supplemental
Experimental Procedures for details.
Cell Culture, Transfection, and Viral Infection
Mouse embryonic fibroblasts (MEFs) and primary hepatocytes were isolated
and cultured following standard protocols. 293FT and HepG2 cells were
cultured in DMEM, supplemented with fetal bovine serum. Plasmid
transfections into 293FT cells and HepG2 were performed with polyethylenimine (PEI; Sigma) and Lipofectamine 2000 (Invitrogen), respectively. siRNA
transfections were performed with Lipofectamine RNAiMAX (Invitrogen).
(B) Quantification of the ratios of PM and IC IR-GFP signal intensities in (A) (mean SD; ****p < 0.0001).
(C) The indicated proteins were incubated for 1 hr and added to beads coupled to the IRMIM-WT peptide. Proteins bound to beads were blotted with the indicated
antibodies. The relative BUBR1 intensities (mean SEM; n = 3 independent experiments) are shown below.
(D) 293T cells were transfected with plasmids encoding MYC-BUBR1, AP2B1, and IR and treated with or without dynasore (Dyn). The total cell lysates (TCL), antiMYC IP, and IgG IP were blotted with the indicated antibodies. The relative intensities of IRb and AP2B1 (mean SEM; n = 3 independent experiments) are shown
in the figure.
(E) HepG2 cells stably expressing IR-WT-GFP were transfected with the indicated siRNAs and stained with anti-GFP (IR; green) and anti-AP2B1 (red) antibodies.
Two boxed regions (1 and 2) were magnified and shown. Scale bar, 10 mm.
(F) Quantification of the Manders coefficients of IR and AP2B1 co-localization in (E) (mean SEM; siLuc, n = 16; si-p31, n = 26; si-p31/siBUBR1, n = 12; si-p31/
siMAD2, n = 11; **p < 0.005).
(G) Western blot analysis of cell lysates in (E) and (F).
See also Figure S4.
SUPPLEMENTAL INFORMATION
AUTHOR CONTRIBUTIONS
E.C. designed and performed all the experiments, analyzed the data, and
wrote the paper. X.Z. and C.X. analyzed copy number variation in hepatocytes.
H.Y. supervised the project, provided suggestions, and edited the paper.
ACKNOWLEDGMENTS
We thank Xuemin Zhang for assistance with generating the p31F/F mice, Jan
van Deursen for providing the Bub1bH/H mice and other reagents, Mayuko
Hara for performing ITC, Bing Li for preparing BUBR1N, Min Kim for advice
on adenovirus production, Ralph DeBerardinis for providing 293FT and
HepG2 cells, Vanessa Schmid and Rachel Bruce for single-cell sequencing,
John Shelton and James Richardson for advice on histological analysis, and
Xuelian Luo for advice on protein purification. We are grateful to anonymous
reviewers for suggesting the genetic suppression experiments. This work is
supported, in part, by grants from the Clayton Foundation (to H.Y.) and the
NIH (UL1TR001105 to C.X.). H.Y. is an HHMI Investigator.
Received: March 23, 2015
Revised: December 9, 2015
Accepted: May 24, 2016
Published: June 30, 2016
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Article
Authors
Steffen Meyer, Martin Woodward,
Christina Hertel, ..., Part Peterson,
Kai Kisand, Adrian Hayday
Correspondence
kai.kisand@ut.ee (K.K.),
adrian.hayday@kcl.ac.uk (A.H.)
In Brief
Self-reactive antibodies specific for type I
interferons are associated with protection
against type I diabetes in patients with an
autoimmune syndrome caused by
mutations in AIRE.
Highlights
d
Article
AIRE-Deficient Patients Harbor Unique
High-Affinity Disease-Ameliorating Autoantibodies
Steffen Meyer,1,11 Martin Woodward,2,11 Christina Hertel,1,11 Philip Vlaicu,1,11 Yasmin Haque,2,11 Jaanika Karner,3,11
Annalisa Macagno,4 Shimobi C. Onuoha,4 Dmytro Fishman,5,6 Hedi Peterson,5,6 Kaja Metskula,7 Raivo Uibo,7 Kirsi Jantti,8
Kati Hokynar,8 Anette S.B. Wolff,9 APECED patient collaborative, Kai Krohn,8 Annamari Ranki,10 Part Peterson,3
Kai Kisand,3,* and Adrian Hayday2,*
1ImmunoQure
SUMMARY
(TSAs) by medullary thymic epithelial cells (mTEC) is directly promoted by AIRE, a poorly understood transcriptional regulator
(Mathis and Benoist, 2009; Klein et al., 2014). Reflecting its
importance, AIRE deficiency is defined by the APS1/APECED
syndrome for which autoimmune polyendocrinopathy and
chronic mucocutaneous candidiasis are pathognomonic (Nagamine et al., 1997).
There are also several mechanisms of peripheral T cell tolerance, including requirements for co-stimulatory signals for the
activation of naive T cells; the expression of molecular brakes
(e.g., CTLA-4, PD-1) by activated T cells; and the suppression of
effector T cells in trans by FOXP3-expressing T-regulatory
(T-reg) cells. Reflecting its importance, FOXP3 deficiency is
defined by early-onset, life-threatening autoimmunity (Bennett
et al., 2001; Wildin et al., 2001).
Central and peripheral tolerance mechanisms have likewise
been hypothesized to shape the B cell compartment. Thus,
self-reactive B cells developing in the bone marrow may be
censored by clonal deletion, clonal anergy, or B cell receptor
(BCR) editing in which secondary gene rearrangements replace
the initial BCR with a new specificity (Goodnow et al., 2010; Pillai
et al., 2011; Ubelhart and Jumaa, 2015). Peripheral B cell tolerance is less well characterized, although some checkpoints
have been inferred. For example, immature transitional B cells
recently emigrated from the bone marrow contain many autoreactive and polyreactive cells, whereas there are relatively few
among mature naive B cells, strongly suggesting that tolerance
is imposed as transitional B cells differentiate into naive B cells
(Wardemann et al., 2003).
Interestingly, this B cell checkpoint is T cell dependent, as reflected by its impairment in patients with T-reg deficiencies
(Kinnunen et al., 2013). Likewise, CD40L and MHC class II
deficiencies that each impair T-B interactions also display
more autoreactive B cells (Meffre and Wardemann, 2008). These
582 Cell 166, 582595, July 28, 2016 2016 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
RESULTS
High-Titer Autoreactivities in APS1/APECED
Sera from 81 APS1/APECED patients from discrete Finnish, Norwegian, Slovenian, and Sardinian cohorts were directed against
a ProtoArray displaying 9000 immobilized recombinant human
proteins or protein fragments. Because some patients were
sampled longitudinally, 97 sera were assayed in total. Control
sera were from healthy first-degree relatives (n = 9) and healthy
unrelated volunteers (n = 12) across the same age range. Data
readouts for the binding of individual sera were normalized by
applying robust linear modeling (Sboner et al., 2009), whereafter
each signal was assigned a Z score denoting the number of standard deviations (SD) above or below the mean of the combined
healthy relatives and controls.
Most patients and the combined controls displayed Z scores
of 12 for 200 proteins (Figure 1A). However, when the convention was employed of defining Z R 3 as bona fide positives, the
patients segregated from the two control cohorts, considered
either jointly or separately. Thus, each control serum displayed
reactivities of Z R 3 toward an average of %20 proteins, with
most recognizing < 10 (Figures 1A, 1B, and S1A). Given that
there was inter-individual variation, the 21 control sera collectively displayed Z R 3 reactivities toward 406 distinct proteins,
i.e., 5% of those displayed on the array (Figure 1B). For only
2 proteins was Z R 4, and for none was Z R 5 (Figures 1A and
1B). Hence, as expected, the control cohorts largely lacked
high-titer serum autoreactivities.
Conversely, most patients at any one time displayed Z R 3
autoreactivities toward R 80 proteins (Figures 1A, 1B, and
S1A). These data were re-analyzed with stringent procedures
to minimize false-positives, including exclusion of any signals
that might have arisen from cross-sample print contamination. With this achieved, the patients private autoantibody
repertoires collectively detected 3,731 distinct targets (Figure 1B). Furthermore, almost all patients displayed Z R 4
scores for at least 10 proteins (mean of 30), collectively
recognizing > 1,500 proteins, and > 50% of patients displayed
Z R 5 scores for R 10 proteins (mean of > 12), collectively
recognizing 636 proteins (Figures 1A and 1B). Hence, high-level
reactivity toward multiple self-proteins was a disease-defining
property. This was further illustrated by the qualitative difference in Z score distribution curves for patients versus controls,
which cannot simply be explained by there being 5-fold more
patient sera (Figure 1C). Thus, whereas sampling greater
numbers would likely have increased the protein species detected by control cohorts at Z R 4, it would not bridge the
1,000-fold gap between two proteins detected by 21 control
sera versus > 1,500 proteins detected by 97 patient sera
(Figure 1B).
In sum, 81 different patients collectively displayed strong reactivities to >40% of human proteins arrayed. For most proteins
(blue dots 133731, Figure 1D), reactivities were spread across
the cohort, reflecting high inter-patient variation, whereas 12
proteins (blue dots 112, Figure 1D), including several type I
IFNs, were recognized by > 60% of patients, as reported
(Meager et al., 2006). However, the public specificities were
not enriched among the 126 reactivities shared between patients
and controls at z > 3 (red dots, Figure 1D), emphasizing that their
common autoantigenicity is unique to the patients. Patient autoreactivity frequencies were largely comparable across geographical locations, albeit somewhat less in Norway and Slovenia, and
age ranges (Figures S1B and S1C). Indeed, most anti-IFN autoantibodies of APECED patients were reported to increase early in
life and remain stable thereafter (Meager et al., 2006; Wolff et al.,
2013).
The collective targets of patient antibodies included intracellular, trans-membrane, and secreted proteins. Because many
proteins displayed on the ProtoArray may be denatured, there
may be false-negatives that underestimate patient reactivities
to conformational determinants. Although a detailed analysis of
the types of proteins targeted will be presented, it is evident
that the proteins most commonly detected by patient sera
included numerous cytokines, particularly type I IFNs, for which
reason this study focuses on the nature of those autoreactivities.
Strong, Selective Anti-Cytokine Reactivities
Human type I IFN genes include 13 IFNa genes, 1 IFNb gene, and
1 IFNu gene. There is also a type II IFNg gene and three type III
IFNl genes. IFNg is largely limited to lymphocytes, whereas
type I and type III IFNs are broadly expressed, with their functional uniqueness and/or redundancy unresolved (Ivashkiv and
Donlin, 2014). As assessed by ProtoArray, patient sera showed
significantly stronger reactivities than controls toward all IFNa
subtypes, albeit the reactivities to some (e.g., a1/13, a5, and
a14) were higher than those to others (e.g., a2, a16, and a21)
(Figure 2A). The differential between patients versus controls
was emphasized by luciferase-based immunoprecipitation
(LIPS) in which many target proteins were recognized in their
native conformations (Figure 2B). Many patients showed strong
reactivities to IFNu but rarely toward IFNb (Figure 2B) and never
toward IFNk and IFN, two phylogenetically distant type I IFNs
(data not shown). By contrast, patient sera harbored reactivities
significantly above controls toward IL1a, IL5, IL6, IL17A, IL17F,
IL20, IL22, IL28A (IFNl2), IL28B (IFNl3), and IL29 (IFNl1) (Figure 2C). Whereas reactivities toward some targets (e.g., IL17F,
IL22) were common to most patients, reactivities toward others
(e.g., IL20, IL28, IL6) were not (Table S1), and with the exception
of IL5, patient sera mostly did not detect either Th2 cytokines
(e.g., IL4 and IL13) or IL21, a Tfh (T follicular helper) cell cytokine
that drives high-affinity antibody maturation. There were also no
reactivities toward G-CSF and GM-CSF (Table S1), which drive
the development of myeloid cells associated with the patients
inflammatory endocrinopathies.
Cytokine reactivities were largely validated by ELISA, which
confirmed that IFNg was only rarely and weakly recognized by
patient sera (Figure 2D; Table S1) and that there was no reactivity
toward TNFa (data not shown). By contrast, ELISA revealed au-
10.86 ng/ml, respectively, were displayed by in-house-generated recombinant sifalimumab and rontalizumab, two anti-IFN
mAbs used in clinical trials for systemic lupus erythematosus patients (Table S4).
Predictably, the antibodies varied in their inhibition of IFNstimulated responses. Thus, 26B9 neutralized IFNu, but not
IFNa16, and only poorly inhibited IFNa8 (Figure 4A; Table S4).
Likewise, in the same assay, rontalizumab failed to efficiently
neutralize IFNa6, IFNa7 and IFNa10, whereas sifalimumab
neutralized several IFNa subtypes only weakly. By contrast,
19D11 neutralized all 12 IFNa subtypes tested (Table S4).
Patient-derived IFN-specific mAbs were also assessed for
their capacity to inhibit STAT1 phosphorylation in cells treated
with each of 12 IFNa subtypes, IFNu, IFNb, or IFNg (Figures
4B, 4C, and 4D). As predicted from the luciferase assay,
19D11 inhibited STAT1 phosphorylation levels (normalized to
total STAT1 or tubulin) driven by all IFNa subtypes but did not
affect responses to IFNu, IFNb, or IFNg. By contrast, 25C3, an
additional patient-derived mAb (Table S2), was highly selective
for discrete IFNa subtypes, whereas other antibodies tested,
including 26B9, showed neutralization profiles between those
of 19D11 and 25C3 (Figures 4B4D). Only 26B9 and 31B4
neutralized IFNu, and none neutralized IFNb or IFNg. By comparison, sifalimumab, rontalizumab, and AGS-009 (another
IFNa-targeting mAb in clinical development) showed variable
and less uniform inhibition of STAT1 phosphorylation induced
by different IFNa subtypes (Figure S4A).
The striking biological activities of patient mAbs were not
limited to those specific for IFNs in that potent functional target
neutralization was shown by mAbs targeting IL17F, IL22,
IL32g, and IL20, respectively (Figure S4B).
Biologically Active Human Antibodies In Vivo
We next asked whether patient autoantibodies could functionally
neutralize targets in vivo. To test this, mice were treated intraperitoneally (i.p.) with a single aliquot of antibodies 26B9, 19D11, or
sifalimumab, and their ears inoculated intradermally (i.d.) on
days 1, 3, 6, and 8 with recombinant human IFNa5 or IFNa14
(Figure 5A) and IFNu (data not shown). Relative to repeated inoculation with vehicle/PBS, the cytokines induced ear swelling, reflecting an inflammatory response that includes rapid TNFa and
IFNg induction (Figures S5A and S5B). This ear swelling was
significantly inhibited by single injections of antibodies (Figure 5B). Again, neutralization varied toward the effector IFNa
subtype: 26B9 and 19D11, but not sifalimumab, largely ablated
the IFNa5 response, whereas all three partially yet significantly
limited swelling induced by IFNa14 (Figure 5B).
Specific, antibody-mediated neutralization in vivo was likewise seen when the same assay was applied to human IL17F
or IL32g (Figures 6A and 6B). For IL17 neutralization, the data are
clearly consistent with the known capacity of APS1/APECED
patients antibodies to neutralize Th17-family cytokines (Kisand
et al., 2010; Puel et al., 2010), thereby predisposing to Candida
infection.
Additionally, the detection of mouse IL22 by antibody 30G1
offered an opportunity to measure its bio-activity toward endogenous IL22, a primary effector of imiquimod (IMQ)-induced
dermatitis used to model psoriasis (van der Fits et al., 2009).
IMQ-induced pathology measured by modified PASI scoring
was significantly ameliorated by 30G1 relative to IgG control,
particularly following an initial inflammatory response (Figures
6C and S6). Again, 30G1 was at least as effective as an inhouse-expressed anti-IL22 antibody, fezakinumab (see above)
(Figure 6C). Collectively these data establish the capacity of
patient anti-cytokine antibodies to limit pathologies induced by
their targets in vivo.
DISCUSSION
This analysis of the impact of AIRE deficiency on human B cells has revealed
a signature pattern of humoral autoreactivity with general implications for our
understanding of autoimmunity. First,
the autoantibodies studied were mostly
extremely high affinity and specific for
native conformational epitopes. These
properties were shared by antibodies
specific for cytokines targeted by most patients (e.g., IFNa,
IL17, IL22) and by antibodies specific for IL20 to which few
patients displayed reactivity. Because such properties are
very rare among antibodies raised by immunization, when B
cells are primed de novo to antigen for short periods of time,
it seems inappropriate to continue to model one type of mAb
on the other.
Second, essentially all 81 APS1/APECED patients studied
showed strong reactivities toward a common set of 1015 proteins, coupled with patient-specific reactivity profiles toward
8090 additional proteins. This limited frequency (< 1% of proteins displayed on the array) is consistent with a recent report
that B cell tolerance was not globally disrupted in 51 APS1/
APECED patients sampled (Landegren et al., 2016). Nonetheless, the patient-to-patient variation in reactivity profiles meant
that the 97 sera analyzed in our study collectively harbored antibodies toward over 3,500 proteins.
The patient-to-patient variation argues that B cell autoimmunity resulting from AIRE deficiency is not simply an amplification
of sporadic, low-level autoreactivities seen in healthy controls
but has distinct origins. By this perspective, defects in central
T cell tolerance may underpin other autoimmune and autoinflammatory pathologies attributed to high-affinity autoantibodies.
Whereas this contrasts with the widely held view that autoimmune diseases mostly reflect peripheral tolerance defects, it
aligns with data that central tolerance defects contribute to the
NOD mouse model of T1D (Geng et al., 1998; Zucchelli et al.,
2005). Moreover, wherever autoantibodies reflect central T cell
tolerance defects, donor-to-donor variation is to be expected,
as individuals will generate distinct TCR repertoires via quasirandom gene rearrangements, will be exposed to different physiologic and environmental triggers that promote the selective
outgrowth of autoreactive T cell clones, and will differ in immune
response modifier genes (e.g., HLA) that regulate the magnitude
of antigen-specific responses.
Autoantibodies to some non-tissue-restricted antigens, including multiple type I IFNa subtypes, are displayed by almost
all patients, sometimes early post-partum (Wolff et al., 2013).
Most likely, the immunogenicity of these proteins arises by
mechanisms distinct from those shaping patient-specific autoantibody repertoires. Possibly the public autoantibodies arise
from a direct impact of AIRE deficiency on B cell tolerance,
for example, via the dysregulation of AIRE-expressing thymic
B cells that resemble GC B cells by several criteria (Yamano
et al., 2015). Arguing against this, however, autoantibodies to
type I IFNs, Th17 cytokines, and additional self-proteins are
found in thymoma patients with AIRE-sufficient B cells (Kisand
et al., 2011; Meager et al., 1997; Wolff et al., 2014). This likewise
argues against autoantibodies to type I IFNs and Th17 cytokines
originating from defects in lymph node AIRE+ cells termed
eTACs (Gardner et al., 2008). Although studies in mice have
suggested tolerizing roles of eTACs, the functions of their rare
human counterparts are unknown (Poliani et al., 2010).
AIRE deficiency may, however, act indirectly on thymic B cells,
for example by hyperactivity of functionally competent thymic gd
cells (Ribot et al., 2009) that may likewise be dysregulated in thymoma. Such cells may create an intra-thymic milieu favoring
priming rather than tolerance of thymic B cells toward proteins
highly expressed in the thymus (Dudakov et al., 2012; Meager
et al., 2006).
Notwithstanding this possibility, our findings suggest that
high-affinity autoantibodies in APS1/APECED patients prob-
That almost all APS1/APECED-derived mAbs were biologically active in vivo against a range of cytokine targets has
profound implications for patients. Clearly, immune-effector responses may be reduced, as in the association of anti-IL22
with susceptibility to Candidiasis (Kisand et al., 2010). Likewise,
gut barrier integrity may be compromised, leading to increased
levels of anti-commensal antibodies (Hetemaki et al., 2016).
Conversely, despite the common neutralization of IFNa and
IFNu, APS1/APECED patients do not show severe viral infections, as were recently reported for a child genetically impaired
in type I IFN (Ciancanelli et al., 2015). Possibly preserved
IFNb function mediates anti-viral protection in APS1/APECED
patients.
On the other hand, some autoantibodies may target key
mediators of immunopathologies, thereby ameliorating disease.
Thus, a unique correlation was observed between antibodymediated neutralization of IFNa and failure to develop T1D,
providing a novel strand of support for animal studies arguing
that targeting type I IFNs could be effective in T1D. The concept
that naturally arising autoantibodies may be beneficial is not
widely considered, despite its underpinning the widespread
592 Cell 166, 582595, July 28, 2016
whereafter values were log-transformed and subjected to robust linear normalization (Sboner et al., 2009). Z scores were calculated as the number of standard deviations of the signal from the mean of the corresponding controls
and healthy relatives; Z R 3 was considered positive. After scoring, stringent
quality assessment was undertaken, including high correlation coefficients of
duplicate spots of printed proteins (average r = 0.92), reactivity toward known
autoantibody targets, and perfect correlation of signals for proteins spotted in
different locations. Printing contaminants were identified as proteins showing
high correlation coefficients with known APECED antibody targets and were
further verified by cross-reference to another protoarray (5.0) used for 23 patients and 7 controls. Thus, 31 suspect false-positives were identified and
excluded from further consideration.
Antibody Isolation and Cloning
Cloning, production, and purification of human mAbs were performed as
described (patent application WO2013/098419). In brief, memory B cells
(CD22+, IgD , IgM , CD3 , CD8 , and CD54 ) were flow-sorted (MoFlo)
from patient PBMC, incubated transiently with EBV-containing B95-8 supernatant (SN) for 3.5 hr at 37 C, and then incubated in Transferrin- and CpGsupplemented IMDM at 37 C, 5% CO2, at 10 cells/well in 96-well plates
coated with irradiated PBMC feeders. Short-term, oligoclonal B cell culture
SN were analyzed for IgG and antigen-specific antibodies detected by
ELISA and/or LIPS. Positive wells were harvested, cells single-cell-sorted
into reverse transcriptase (RT) buffer (Life Technologies), and RT-PCR
performed using Superscript III (Life Technologies) and random hexamers.
IgG VH, Vl, and Vk regions were amplified from cDNA by two-step nested
PCR reaction using Advantage 2 cDNA polymerase (Clontech) and primer
mixes specific for germline families (VBASE database). Nested primers
attached restriction sites for V-region cloning into expression vectors
providing IgG1, Ig-k, or Ig-l constant regions. Recombinant antibodies were
produced in HEK293T cells and antigen specificity analyzed by ELISA. Corresponding closest germline region sequences were identified using the
VBASE2 database (Retter et al., 2005). CDRs were identified by IMGT definitions (Lefranc, 2003).
Complete Ig-VH and VL regions described in US7741449 (Sifalimumab),
US7087726 B2 (Rontalizumab), US8361463 (ACO-1), and US20070258982
A1 (Fezakinumab) were ordered as CHO-codon-optimized synthetic constructs (GenScript) and expressed as above.
mAb Characterization In Vitro
EC50 binding of mAbs was determined by ELISA. Neutralizing capacities of
type I IFN-specific mAbs were studied using phospho-STAT1 quantification
in immunoblot and ISRE-luciferase reporter assay. IL17F, IL22, IL20, and
IL32 neutralization assays were performed on respective responsive cell lines.
mAB affinities were measured with a Biacore T200 (GE Healthcare). Epitope
mapping used overlapping 18-mer peptides.
mAb Characterization In Vivo
C57BL/6J (WT; from Charles River) mice were administered i.p. with mAbs
(day 0) and inoculated i.d. on days 1, 3, 6, and 8 with cognate human cytokines,
IFNa2a, IFNa2b, IFNa4, IFNa14, IL17F, and IL32g, and their ear thicknesses
measured with a micrometer. For IL22 mAbs cross-reactive to mouse, bioactivity was assessed in imiquimod-treated mice.
SUPPLEMENTAL INFORMATION
AUTHOR CONTRIBUTIONS
S.M., A.M., and S.C.O. cloned monoclonal antibodies from patient samples,
and K.J. and K.H. assisted. S.M., P.V., and A.M. characterized antibodies
in vitro; M.W. and Y.H. did so in vivo. C.H. analyzed ProtoArray data and wrote
and edited the paper. J.K. assayed neutralization by sera and Tfh subsets and
performed LIPS. D.F. and H.P. analyzed ProtoArray data. K.M. and R.U.
screened sera for T1D autoantibodies and tested germline antibody specificities. K. Krohn and A.R. developed the clinical database, sampled Finnish
patients, and employed ELISA. A.S.B.W. sampled Norwegian patients,
contributed to the clinical database, and assayed antibodies. APECED patient
collaborative contributed to the clinical database and sampled respective patients. P.P., K. Kisand, and A.H. supervised research, reviewed data, and
wrote and edited the paper.
ACKNOWLEDGMENTS
We are indebted to patients; to the Finnish APECED and Addison patients
association; and to attending physicians and carers. We thank M. Rothe,
P. Adler, A. Remm, M. Pihlap, M. Karlsberg, A. Tallqvist, M. Tuukkanen,
L. Prassmayer, M. Wordehoff, A. Peters, R. Repke, B. Mathis, and particularly
E. Stuart and K. Henco for critical insight and support. We thank staff of the
Biological Services Unit at Kings College London. Funding was by the
following: ImmunoQure AG, the Wellcome Trust, and CRUK (to A.H.) and
Estonian Research Council grant IUT2-2 and European Union Project 20142020.4.01.15-0012 (to J.K., P.P., and K. Kisand). P.P., K. Krohn, K. Kisand,
A.R., and A.H. are cofounders and shareholders of ImmunoQure AG, and
A.M., C.H., P.V., S.C.O., and S.M. were/are employees of ImmunoQure AG.
Received: January 27, 2016
Revised: April 24, 2016
Accepted: June 10, 2016
Published: July 14 2016
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Article
Authors
Nicole L. Kallewaard, Davide Corti,
Patrick J. Collins, ..., Qing Zhu,
Steven J. Gamblin, John J. Skehel
Correspondence
zhuq@medimmune.com (Q.Z.),
john.skehel@crick.ac.uk (J.J.S.)
In Brief
Identification of a human monoclonal
antibody that reacts effectively with all
influenza A hemagglutinin subtypes
paves the way for developing
immunotherapy for people infected with
the flu virus.
Highlights
d
Accession Numbers
5JW5
5JW4
5JW3
KX398429
KX398468
Article
Structure and Function Analysis of an Antibody
Recognizing All Influenza A Subtypes
Nicole L. Kallewaard,1,8 Davide Corti,2,8 Patrick J. Collins,3,8 Ursula Neu,3,8 Josephine M. McAuliffe,1 Ebony Benjamin,1
Leslie Wachter-Rosati,1 Frances J. Palmer-Hill,1 Andy Q. Yuan,4 Philip A. Walker,5 Matthias K. Vorlaender,3 Siro Bianchi,2
Barbara Guarino,2 Anna De Marco,2 Fabrizia Vanzetta,2 Gloria Agatic,2 Mathilde Foglierini,6 Debora Pinna,6
Blanca Fernandez-Rodriguez,6 Alexander Fruehwirth,6 Chiara Silacci,6 Roksana W. Ogrodowicz,5 Stephen R. Martin,5
Federica Sallusto,6 JoAnn A. Suzich,1 Antonio Lanzavecchia,6,7,9 Qing Zhu,1,9,* Steven J. Gamblin,3,9
and John J. Skehel3,9,*
1Department
of Infectious Disease and Vaccines, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA
BioMed SA, Via Mirasole 1, 6500 Bellinzona, Switzerland
3Mill Hill Laboratory, The Francis Crick Institute, London NW7 1AA, UK
4Department of Antibody Discovery and Protein Engineering, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA
5Structural Biology Science Technology Platform, Mill Hill Laboratory, Francis Crick Institute, London NW7 1AA, UK
6Institute for Research in Biomedicine, Universita
` della Svizzera italiana, 6500 Bellinzona, Switzerland
7Institute for Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
8Co-first author
9Co-senior author
*Correspondence: zhuq@medimmune.com (Q.Z.), john.skehel@crick.ac.uk (J.J.S.)
http://dx.doi.org/10.1016/j.cell.2016.05.073
2Humabs
SUMMARY
596 Cell 166, 596608, July 28, 2016 2016 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
RESULTS
Isolation, Genetic Description, and Optimization of
MEDI8852
Four broadly reactive antibodies were isolated from the memory
B cells of a selected donor based on influenza A HA protein
cross-reactivity as previously reported (Traggiai et al., 2004;
Pappas et al., 2014). These antibodies (FY1, FY5, FY6, and
FY18) belong to the same lineage carrying VH6-1*01/D3-3*01/
JH3*02 and VK1-39*01/JK1*01 gene segments (Figure 1A). We
reconstructed the genealogical trees of this lineage and produced the unmutated common ancestor (UCA), the four clonally
derived antibodies, and three antibodies representing the evolutionary branching points (BP) of the lineage (Figure 1B). Purified
antibodies were tested for neutralizing activity against multiple
viruses of different group 1 and 2 subtypes (Figure 1C). Interestingly, the UCA antibody exhibited neutralizing activity against
group 1 viruses, but not group 2 viruses, albeit with lower
potency as compared to some of the mutated antibodies. Of
note, the first BP (BP1) gained neutralization activity toward early
group 2 H3N2 viruses (HK/68 and VC/75). Two antibodies (i.e.,
FY1 and FY5) of this lineage acquired neutralization activity
against group 2 viruses through two independent pathways of
somatic mutations. The same analysis was extended to the lineage of FI6, a previously described antibody cross-neutralizing
group 1 and group 2 viruses (Corti et al., 2011). Isolation of five
additional antibodies from this lineage allowed the reconstruction of a complex genealogy tree (Figure S1). Similarly to what
was observed for the FY1 lineage, the FI6-UCA antibody exhibited neutralizing activity against group 1 viruses only and
evolved through two independent pathways of somatic mutations that led to the group 1-specific FI370 and FI6038 antibodies
and to the group 1 and 2 cross-reactive antibodies FI6, FI2013
and FI4013. Taken together, these findings suggest that in
both lineages, the UCA was initially selected by a group 1 virus
and developed to a branching point characterized by crossreactivity toward a limited number of group 2 viruses. From
this point, the final antibody may have been selected further for
binding to group 1 only (e.g., FY6 and FI370) or group 2 HAs
(e.g., FY1 and FI6). These results are consistent with a model
in which the development of cross-reactive group 1 and 2 antibodies is started by group 1 HAs and then further selected
through boosts by group 2 HAs.
The FY1 antibody was chosen as the lead, based on its potency, breadth, and low somatic mutations for further in vitro
optimization through parsimonious mutagenesis of the complementarity determining regions (CDRs) combined with reversion
of unnecessary somatic mutations in the frameworks. The optimization focusing on affinity binding resulted in a 14-fold and
5-fold improved Fab affinity to H3 HA and H1 HA proteins determined by surface plasmon resonance, respectively (Table S2).
The resulting antibody was named MEDI8852 (VH and VL sequences shown in Figure 1A) and was compared side by side
with the parental FY1 antibody for binding and neutralization of
a large panel of influenza A viruses. MEDI8852 showed higher
binding activity as compared to FY1 against the group 1 HA proteins of H1, H2, H5, H6, and H9 subtypes and group 2 HA proteins of H3 and H7 subtypes, with a mean half-maximal effective
Cell 166, 596608, July 28, 2016 597
FY5
I
.
.
.
.
.
.
.
.
113
107
102
T I F GV N I D A F D I WGQG TMV T V S S
.V. . . . . . . . . . . . . . . . . . . . .
.V. . . . . . . . . . . . . . . . . . . . .
.V. . . . . . . . . . . . . . .K. . . . .
.V. . . . . . . . .V. . . . . . . . . . .
. V . . L . . . . Y . . . . . . AK . . . . .
.V. . . . . . . . . . . .L. .K. . . . .
. V . . . . V . . . .M. . . . . . . . . . .
. V . . . . V . . . .M. . . . . . . . . . .
107
100
96
90
85
80
75
70
100
100A
100B
100C
100D
100E
100F
100G
100H
95
90
85
80
82A
82B
82C
75
70
65
60
65
60
10-1
C
FY5
FY6
FY
6
FY
18
25 (13)
100
17 (6)
FY
5
18 (8)
FY18
FY18
101
FY
1
17 (18)
9 (6)
BP3
15 (12)
BP3
BP2
2 (2)
6 (3)
3 (2)
BP
15 (11)
FY1
BP2
BP
5 (1)
S S L QP E D F A T Y Y CQQS R T F GQG T K V E
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . .V. . . . . . . . . . . . . . . . . .
.N. . . . . . . . . . . .L. . . . . . . . . . .
. TF . A . . V . . . . . . . . . . . . . . . . . .
. . . . . . .V. . . . . .L. . . . .H. . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
102
BP
BP1
2 (1)
I
.
.
.
.
.
.
.
.
UCA
2 (2)
UCA
BP1
55
50
55
50
45
40
35
30
4 (2)
FY1
52A
52B
30
25
15
15
10
5
5
20
I
.
.
.
.
.
.
.
.
IC50 ( g/ml)
I QMT QS P S S L S A S VGDR V T
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . I .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
45
25
D
UCA
.
BP1
.
BP2
.
BP3
.
FY18
.
FY5
.
FY6
.
FY1
MEDI8852 .
40
VL
35
35A
35B
I
.
.
.
.
.
.
.
.
20
UCA
QVQ L QQSGPG L V K P SQ T L S L T C A
BP1
. . . . . . . . . . . . . . . . . . . . . . .
BP2
. . . . . . . . . . . . . . . . . . . . . . .
BP3
. . . . . . . . . . . . . . . . . . . . . . .
FY18
. . . . . . . . . . . . . . . . . . . . . . .
FY5
. . . . . . . . . . . . . . . . . . . . . . .
FY6
. . . . . . . . . . . . . . . . . . . . . .V
FY1
. . . . .E. . . . . . . . . . . . . . . . .
MEDI8852 . . . . . . . . . . . . . . . . . . . . . . .
10
VH
I
.
.
.
.
.
.
.
.
K
.
.
.
.
.
.
.
.
Group 1
Group 2
H1N1 WSN/33
H1N1 PR/34
H1N1 FM/47
H1N1 BJ/95
H1N1 SZ/95
H1N1 NC/99
H1N1 SI/2006
H1N1 SD/2007
H1N1 CA/2009
H1N1 BR/2010
H2N2 JP/57
H5N1 VT/2004
H6N2 AB/85
H9N2 HK/97
H3N2 HK/68
H3N2 VC/75
H3N2 SG/93
H3N2 WH/95
H3N2 SY/97
H3N2 PA/99
H3N2 CA/2004
H3N2 WI/2005
H3N2 PT/2009
H3N2 VC/2011
H7N3 BC/2004
FY6
H8
H4 H14 H3
H12
H10
H7
H9
IC50( g/ml)
H2
H11
H6 H1
0.1
102
101
100
FY1
10-2
H5
10-1
10-1
H15
H16
H13
Group 1
H1
H2
H5
H6
H9
Group 2
H3
H7
EC50( g/ml)
Group 1
100
MEDI8852
H1N1
WSN/33
PR/34
FM/47
NJ/76
KW/86
TX/91
SZ/95
BJ/95
NC/99
SD/2007
SI/2006
CA/2009
BR/2010
HK/2010
NH/2010
NY/2012
WA/2012
BO/2013
H3N2
HK/68
VC/75
LA/87
SG/93
WH/95
SY/97
PA/99
CA/2004
WI/2005
PT/2009
VC/2011
BR/2011
NY/2012
TX/2012
AM/2013
SW/2013
NC/2014
PU/2014
IC50 ( g/ml)
101
100
10-1
CR9114
39.29
FI6v3
10
MEDI8852
104
103
102
101
FY1 MEDI8852
102
10-2
MFI
Group 2
FY1 MEDI8852
Mock
H1 SC/18
H8 ON/68
H11 ME/74
H12 AL/76
H13 MA/77
H16 SW/99
H17 GU/09
H18 PU/10
H4 CZ/56
H10 GE/49
H14 AS/82
H15 AU/79
102
IC50 ( g/ml)
H2N2 JP/57
H2N3 MO/2006
H5N1 HK/2003
H5N1 VT/2004
H6N2 AB/85
H6N1 HK/97
H9N2 HK/97
H9N2 HK/99
H3N2v MN/2010
H3N2v IN/2011
H7N7 NT/2003
H7N3 BC/2004
H7N9 AN/2013
101
100
10-1
H1N1 PR/34
H1N1 SD/2007
H1N1 SI/2006
H1N1 CA/2009
H1N1 HK/2010
H1N1 WA/2012
H1N1 BO/2013
H2N2 JP/57
H2N3 MO/2006
H5N1 HK/2003
H5N1 VT/2004
H6N2 AB/85
H6N1 HK/97
H9N2 HK/97
H9N2 HK/99
FY1 MEDI8852
H3N2 HK/68
H3N2 WI/2005
H3N2 PT/2009
H3N2 BR/2011
H3N2 NY/2012
H3N2 PU/2014
H3N2 SW/2013
H7N7 NT/2003
H7N9 AN/2013
Isotype Control
0 5 10 20 40
MEDI8852
5 10 20 40
100
MEDI8852
H1N1
H3N2
Control Ab
H1N1
H3N2
40
MEDI8852
Control Ab
ADCC (%)
80
CDC (%)
ADCP (%)
H1
delayed until day 4 post infection with WSN/33 H1, or day 3 post
infection with rHK/68 H3 (Figures 4C and 4E). Significant survival
benefits were also seen with 1 and 3 mg/kg doses when administered on days 1, 2, or 3 post infection, albeit lower survival rates
than the 10 mg/kg (Table S3). MEDI8852 treatment of 10 mg/kg
at all times post infection resulted in significantly decreased viral
titers, compared to control antibody treated and untreated
animals, with a clear trend for greater reductions with earlier
treatment (Figures 4D and 4F).
To further investigate MEDI8852s therapeutic potential, we
determined the therapeutic window for treating ferrets infected
with the highly pathogenic avian influenza virus, A/Vietnam/
1204/2004 H5N1 (VT/2004 H5N1). In these studies, ferrets
were infected intranasally with 1 LD90 of VT/2004 and then
treated with a single intravenous (i.v.) dose of 25 mg/kg of
MEDI8852 at 1, 2, or 3 days post infection. We also used as a
comparator, the anti-influenza drug oseltamivir at 25 mg/kg
twice a day (b.i.d.) (Figure 4G). As expected, all control animals
showed signs of infection including fever peaking from days 1
3 post infection and 100% mortality by day 7 post infection (Figures 4G and 4H). In comparison, ferrets treated with MEDI8852
or oseltamivir on day 1 post infection were completely protected.
When treatment was delayed until 2 or 3 days post infection,
MEDI8852 provided complete protection with 100% survival
while oseltamivir only partially protected animals with survival
rates of 71% and 29%, respectively. In addition, MEDI8852
treatment resulted in a period of fever reduction following
administration, which was not observed in oseltamivir or control
antibody-treated animals (Figure 4H). Similar efficacy and therapeutic window results were seen when MEDI8852 and oseltami600 Cell 166, 596608, July 28, 2016
40
20
0
0
WSN/33 H1
Survival (%)
C 100
80
40
20
2
4 6 8 10 12 14
Days Post Infection
rHK/68 H3
Survival (%)
100
80
40
80
60
40
MEDI8852 F
10 mpk
MEDI8852 Oseltamivir
25 mpk 25 mpk (2x)
Day 1
Day 2
Day 3
*
Ctr. mAb
(Day 1)
2 4 6 8 10 12
Days Post Infection
7
6
5
4
Ctl. mAb
Naive
Day 1
Day 2
Day 3
Day 4
D 10
MEDI8852
10 mpk
Day 1
Day 2
Day 3
Day 4
4 6 8 10 12 14
Days Post Infection
20
0
0
Ctl. mAb
Naive
Ctl. mAb
Naive
20
100
A/VT/2004 H5N1
Survival (%)
*
*
*
60
0
0
*
*
*
*
60
0
0
4 6 8 10 12 14
Days Post Infection
Log TCID50 /g
60
Log TCID50 /g
80
8
Log TCID50 /g
MEDI8852 B
Day 0 Tx
3 mpk
*
1 mpk
*
0.3 mpk
*
0.1 mpk
0.03 mpk
CA/2009 H1
Survival (%)
A 100
9
8
7
6
5
4
3
*
*
9
8
7
6
5
4
3
41
40
39
38
37
36
(MEDI8852)
41
40
39
38
37
36
2
3
(Oseltamivir)
1
2
3
4
Days Post Infection
et al., 2009), all recognize helix A of HA2 and the adjacent hydrophobic groove (Figure 7B, blue box). In contrast, the group
2-neutralizing antibodies CR8020 and CR8043 (Ekiert et al.,
2011; Friesen et al., 2014) recognize a different region of the
fusion peptide and a small b sheet below it in the fusion domain
(Figure 7B, red box). The epitope of MEDI8852, uniquely among
the cross-group neutralizing antibodies reported to date, represents a combination of both regions. A structural comparison of
HA-bound MEDI8852 overlapped with the antibodies CR8020
and CR9114 is shown in Figures S7A and S7B, which reveals
that the overall orientation of MEDI8852 is such that it sits slightly
higher on the HA than the CR8020 antibody and lower than
CR9114. The nearest paratope residue of MEDI8852 is 20
from the membrane proximal end of the HA.
MEDI8852 and 39.29 antibodies bind similarly to residues in
the hydrophobic groove and adjacent helix A in the fusion
domain. Both antibodies contain the four amino acid sequence
ValPheGlyVal/Ile in their otherwise dissimilar CDRH3 loops.
These tetrapeptides superpose in the complexes with an allatom root-mean-square deviation (RMSD) of 0.7 A, indicating
that they interact with their cognate HAs in a similar way (Figure 7C). However, other contacts made by these antibodies
with HA are quite different between MEDI8852 and 39.29, reflecting the fact that the two antibodies are not particularly similar
in sequence and are derived from different germline sequences
(VH6-1*01 and VK 1-39*01 for MEDI8852 versus VH3-30*01
and VK3-15*01 for 39.29). MEDI8852 contacts the base of helix
A and the fusion peptide with its CDRL1 and CDRH2 loops,
respectively (Figure 6). By contrast, contacts made by the heavy
chain of 39.29 Fab mainly involve CDRH3. 39.29 appears to bury
helix A using all three light chain CDR loops. As a consequence,
the 39.29-HA interaction buries a total of 2,287 A2, while
MEDI8852 achieves similar affinity with a smaller buried surface
of 1,646 A2.
MEDI8852 is the second cross-group neutralizing antibody for
which structures of complexes with both group1 and group 2
HAs have been reported. The first was FI6v3, which, although
it recognizes both group 1 and group 2 HAs, has higher in vivo
neutralizing activity against group 1 viruses (Corti et al., 2011).
The cross-group binding of FI6v3 has been attributed to its
long and flexible HCDR3, which can accommodate the differences in conformation and environment of HA2 Trp21 observed
between group 1 and group 2 viruses. There is a much more significant rearrangement between FI6v3 bound to H1 HA (group 1)
versus H3 HA (group 2) than there is between MEDI8852 in its
complexes with H5 HA (group 1) and H7 HA (group 2) which overlap very closely (Figures 5B and S7C). MEDI8852, therefore,
binds in a very similar way to HAs of both groups. This greater
structural conservation of the binding interface is likely responsible for its broader neutralizing ability.
DISCUSSION
There is an unmet medical need for effective treatments against
severe influenza. The potential of broadly infectivity-neutralizing
antibodies used therapeutically to address this need has provided a stimulus for their isolation and characterization. Among
the antibodies considered to date, the anti-HA human monoclonal antibody MEDI8852 has demonstrated significant breadth
of its infectivity-neutralizing capacity. MEDI8852 reacts with HAs
of all influenza antigenic subtypes, potently neutralizes diverse
virus strains with numerous HA subtypes, and can block infection and lethality caused by influenza viruses when administered
up to 4 days after challenge with the virus in mice and up to
3 days post challenge in ferrets with the highly pathogenic
H5N1 virus. This potential ability to overcome the unpredictable
characteristics of influenza, namely the antigenic shift, that results in disease during pandemic periods, and the antigenic drift,
that occurs with the emergence of antigenically novel viruses, is
a major advantage for a candidate anti-influenza therapeutic
antibody.
The mechanisms of MEDI8852-mediated neutralization of
infection involve processes at the beginning and the end of the
infection cycle. Binding of the antibody to HAs on the infecting
virus inhibits HA-mediated membrane fusion that is required
for the initiation of infection. At the end of infection, antibody
binding to precursor HA0 can block its cleavage and prevent
the formation and spread of newly made infectious virus. Additionally, binding of MEDI8852 to HAs displayed on the surfaces
of infected cells results in their recognition and lysis by other
components of the immune system: NK cells, macrophages,
and complement. These multiple mechanisms exhibited by
MEDI8852 presumably combine to ensure the observed effectiveness of antibody treatments in infected mice and ferrets.
The epitope recognized by MEDI8852 is consistent with the
ways it blocks HA function in membrane fusion and with the
locations of epitopes that have been described previously for
influenza group-specific cross-reactive antibodies and for
more broadly reactive antibodies. However, the regions of HA
that interact with MEDI8852 are a combination of those previously assigned to group 1 specificity (primarily a hydrophobic
groove and the adjacent helix A of HA) (Ekiert et al., 2009; Sui
et al., 2009) or group 2 specificity (a separate part of the fusion
peptide, near its N terminus) (Ekiert et al., 2011; Friesen et al.,
2014). The structural characterization of MEDI8852 bound and
unbound structures also highlight the coordinated movement
of the CDRH3 and the CDRL1 to insert into the hydrophobic
grove of the HA, as well as the rearrangement of the orientation
of the glycan attached to Asn38 of the H7 virus to allow antibody
binding. Importantly, structures of the complexes formed by
MEDI8852 with H5 and H7 HAs indicate that the locations and
(E) Location of mutations found during affinity maturation of FY1 to MEDI8852. The variable domains of MEDI8852 are shown in cartoon representation, viewed
from the direction of HA. Regions of the heavy and light chains in contact with HA are colored orange and green, respectively. Interacting sidechains are shown in
stick representation. Residues that differ between the parental and affinity-maturated antibody are shown in sphere representation.
(F) Sequences of MEDI8852 variable region framework and CDR residues. CDRs (according to Kabat) are highlighted in orange and green for the heavy and light
chains respectively. Residues in contact with HA are colored red and residues changed during affinity maturation from FY1 are colored cyan, with corresponding
residues of FY1 indicated.
See also Figure S6.
Figure 7. MEDI8852 Binds to a Unique Site within the H5 and H7 HA Proteins through CDR-H3 and CDR-L1 Conformational Rearrangements
upon Complex Formation
(A) Conformational rearrangements in MEDI8852 on complex formation. Conformational change of the CDRH3 and CDR-L1 loops upon HA engagement. The apo
structure of MEDI8852 is shown in blue, the bound structure is shown in orange and green for the heavy and light chains, respectively. The beginning and end of
the moving regions are indicated with black ovals. HA (H7) is shown as a gray surface. The apo structure does not make interactions with HA and does not fit into
its surface featuresthe conformational change is necessary for productive HA engagement.
(B) Epitopes of different broadly neutralizing antibodies on the HA surface. Residues of HA that are in contact with the heavy chain are colored orange, residues
that are in contact with the light chain are colored green, and residues that are in contact with both chains are colored yellow. The blue box encases the part of the
MEDI8852 epitope (helix A and hydrophobic groove) that can also be found in other broadly neutralizing antibodies as well as group 1 specific ones. The red box
encases the part of the MEDI8852 epitope that can also be found in group 2 specific antibodies (middle of fusion peptide).
(C) Comparison of the structures of the conserved CDRH3 tetra-peptide in the complexes between MEDI8852 and H7 HA (left panel) and 39.29 and H3 HA (right
panel). In both cases the tetra-peptide is shown in stick representation with other loops of the antibody shown as coil, colored as in panel A. The HAs are shown in
surface representation.
See also Figure S7.
ACCESSION NUMBERS
SUPPLEMENTAL INFORMATION
The accession number for the coordinates and structure factors reported in
this paper is PDB: 5JW5 (apo MEDI8852, 5JW4 (H5 complex), and 5JW3
(H7 complex). The accession number for the sequences for all of the antibodies reported in this paper is GenBank: KX398429-KX398468.
elements, produced figures, and carried out bioinformatic analysis. D.P. and
C.S. carried out donors selection and screenings for the identification of
cross-reactive antibodies. F.V. and A.F. carried out cloning HAs and testing
antibodies for binding in cytofluorimetry. S.B. performed biochemical and
cellular assays to test the fusion and HA maturation inhibiting activities of isolated antibodies. B.G. and A.D.M. carried out ADCC and CDC studies. F.S.
wrote the paper. A.L. and D.C. directed the B cell isolation studies, analyzed
data, and wrote the paper. J.M.M. and E.B. carried out in vivo studies. L.W.-R.
carried out ADCP studies. F.J.P.-H. carried out the ELISA binding studies.
N.L.K., Q.Z., L.W.-R., and F.J.P.-H. carried out the neutralization studies.
A.Q.Y. lead the antibody optimization. J.A.S. edited the paper and provided
supervision. N.L.K. and Q.Z. directed antibody optimization, in vitro and in vivo
characterization, analyzed the data, and wrote the paper. P.J.C., U.N.,
P.A.W., M.K.V., R.W.O., S.R.M., S.J.G., and J.J.S. designed and performed
structural research, contributed new reagents and analytical tools, analyzed
data, and wrote the paper.
CONFLICTS OF INTEREST
A.L. is the scientific founder of Humabs BioMed SA. A.L. holds shares in Humabs BioMed. B.G., A.D.M., G.A., F.V., and D.C. are employees of Humabs
Biomed. This work was funded by MedImmune, LLC, a wholly owned subsidiary of AstraZeneca Pharmaceuticals. N.L.K., J.M.M., E.B., L.W.-R., F.J.P.-H.,
A.Q.Y., J.A.S., and Q.Z. were employed by MedImmune, LLC when work was
executed and may currently hold AstraZeneca stock or stock options.
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Article
Authors
M. Gordon Joyce, Adam K. Wheatley,
Paul V. Thomas, ..., Peter D. Kwong,
John R. Mascola, Adrian B. McDermott
Correspondence
pdkwong@nih.gov (P.D.K.),
jmascola@nih.gov (J.R.M.),
adrian.mcdermott@nih.gov (A.B.M.)
In Brief
Quantifying B cells capable of producing
broadly neutralizing antibodies against
influenza serves as a metric to guide the
development of a universal influenza
vaccine.
Highlights
d
Accession Numbers
5K9J
5K9K
5K9O
5K9Q
5KAN
5KAQ
KX386124KX387227
Article
Vaccine-Induced Antibodies that Neutralize
Group 1 and Group 2 Influenza A Viruses
M. Gordon Joyce,1,8 Adam K. Wheatley,1,8 Paul V. Thomas,1,8 Gwo-Yu Chuang,1,8 Cinque Soto,1,8 Robert T. Bailer,1
Aliaksandr Druz,1 Ivelin S. Georgiev,1,2,3 Rebecca A. Gillespie,1 Masaru Kanekiyo,1 Wing-Pui Kong,1 Kwanyee Leung,1
Sandeep N. Narpala,1 Madhu S. Prabhakaran,1 Eun Sung Yang,1 Baoshan Zhang,1 Yi Zhang,1 Mangaiarkarasi Asokan,1
Jeffrey C. Boyington,1 Tatsiana Bylund,1 Sam Darko,1 Christopher R. Lees,1 Amy Ransier,1 Chen-Hsiang Shen,1
Lingshu Wang,1 James R. Whittle,1 Xueling Wu,1 Hadi M. Yassine,1 Celia Santos,1,4 Yumiko Matsuoka,4
Yaroslav Tsybovsky,5 Ulrich Baxa,5 NISC Comparative Sequencing Program,6 James C. Mullikin,6 Kanta Subbarao,4
Daniel C. Douek,1 Barney S. Graham,1 Richard A. Koup,1 Julie E. Ledgerwood,1 Mario Roederer,1 Lawrence Shapiro,1,7
Peter D. Kwong,1,* John R. Mascola,1,* and Adrian B. McDermott1,*
1Vaccine
Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
of Pathology, Microbiology, and Immunology and Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville,
TN 37232, USA
3Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, TN 37232, USA
4Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892, USA
5Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Frederick National Laboratory for
Cancer Research, Frederick, MD 21702, USA
6NIH Intramural Sequencing Center (NISC), National Human Genome Research Institute, National Institutes of Health, Bethesda,
MD 20892, USA
7Department of Biochemistry & Molecular Biophysics and Department of Systems Biology, Columbia University, New York, NY 10027, USA
8Co-first author
*Correspondence: pdkwong@nih.gov (P.D.K.), jmascola@nih.gov (J.R.M.), adrian.mcdermott@nih.gov (A.B.M.)
http://dx.doi.org/10.1016/j.cell.2016.06.043
2Department
SUMMARY
Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/
MIV-prime-boost influenza vaccine trial, we sorted
hemagglutinin cross-reactive memory B cells and
identified three antibody classes, each capable of
neutralizing diverse subtypes of group 1 and group
2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs
to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences
from at least one multidonor class, andin half the
subjectsmultidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast,
these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency
of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide
universal influenza A immunization strategies.
INTRODUCTION
Influenza A viruses can be categorized into two phylogenetic
groups (group 1 and group 2), each containing diverse subtypes
(Figure 1A). Currently, group 1 influenza viruses from the H1 subtype (1918 and 2009 H1N1 pandemics), and the group 2 H3 subtype (1968 H3N2 pandemic), co-circulate and cause seasonal infections in over 10% of the human population each year. Other
subtypes have emerged or threaten to re-emerge including the
group 1 H2 subtype, endemic in humans from 19571968, the
group 1 H5 subtype, which includes lethal avian strains (Subbarao et al., 1998), and the group 1 H6 and H9 and the group 2
H7 and H10 subtypes, which have been associated with human
infections and fatalities in recent years (Butt et al., 2005; Morens
et al., 2013). Frequent zoonotic cross-overs that may cause pandemics of unpredictable frequency and severity highlight the
need for a universal influenza vaccine that is capable of eliciting
protection against divergent influenza A viruses.
Potential approaches to a universal influenza vaccine involve
the elicitation of neutralizing antibodies that recognize the
influenza hemagglutinin (HA) from multiple subtypes. One means
to accomplish this involves ontogeny-based strategies, which
seek to identify antibodies of reproducible classes and to induce
similar antibodies by vaccination (Jardine et al., 2015; Lingwood
et al., 2012; Pappas et al., 2014). We consider antibodies to be of
the same class when they recognize the same region, employ the
same structural mode of recognition, and develop through
similar recombination and maturation pathways (Kwong and
Mascola, 2012). Reproducible classes, which are observed in
multiple individuals, represent immunological solutions to the
challenge of broad influenza A neutralization that might be available to the general human population.
The influenza A-neutralizing stem-directed antibodies that utilize the HV1-69 germline gene are one such multidonor class
(Ekiert et al., 2009; Kashyap et al., 2010; Sui et al., 2009; Throsby
Cell 166, 609623, July 28, 2016 Published by Elsevier Inc. 609
B
H12
H8
H7
H15
H10
H1
H4
H14
H18
H2
H17
H3
H5
H
5
10 5
10 4
01
16
31
36
54
56
10 4
10 3
10 2
10 3
10
27
29
10 2
10 2
59
10 1
10 1
10 1
H5N1
Group 1
Subject 16
Subject 31
0.08%
0.22%
0.07%
Subject 36
Subject 54
Subject 56
10 3
H3-A/Perth/16/2009
H11
Subject 01
H9
H6
H13 H16
H3N2
H7N7
Group 2 D
0.2
Subject 01
Subject 16
H5-A/Indonesia/5/2005
Subject 31
E
Number of
genetic
Similarities
Antibody name
(subject.lineage.clone)
235
136
HV4-34
HV1-18
HV3-23
Subject 54
Subject 36
Subject 56
HV6-1
HV1-18
HV6-1
HV
1-18
94
0.13%
0.29%
0.11%
102
13
HV 4-30
HV1-18
65
HV6-1
HV3-64D
Neutralization breadth
Frequentist
probability
Number of
clones per
subject per
genetic cluster
HV
gene
HD
gene
HJ
gene
CDR H3
HV
length* maturation
LV
gene
LJ
Gene
CDR L3
length*
Binding
competition
Group 1
Group 2
31.g.01
54.f.01
56.a.09
4.9E-12
1
1
30
HV6-1
HV6-1
HV6-1
HD3-3
HD3-3
HD3-3
HJ4/5
HJ4/5
HJ4/5
16
16
16
8%
6%
4%
KV3-20
KV3-20
KV3-20
KJ2
KJ3
KJ2
9
9
9
n.d.
+++
+++
n.d.
H1, H2, H5
H1, H5
n.d.
H3, H7
H3, H7
01.k.01
31.b.09
3.6E-6
1
26
HV1-18
HV1-18
HD3-9
HD3-9
HJ4
HJ4
15
15
9%
5%
KV2-30
KV2-30
KJ5
KJ2
9
9
+++
+++
H1, H5
H1, H5
H3
H3, H7
16.g.07
54.a.84
6.9E-5
8
92
HV1-18
HV1-18
HD2-15
HD2-15
HJ2
HJ2
21
21
11%
11%
KV1-12
KV3-11
KJ2
KJ1
9
9
+++
+++
H1, H5, H9
H1, H5, H9
H3, H7
H3, H7
H3, H7
16.a.26
93
HV1-18
HD2-2
HJ5
21
8%
KV1-39
KJ2
+++
H1, H5, H9
54.a.39
92
HV1-18
HD2-2
HJ5
21
6%
KV3-11
KJ1
+++
H1, H9
H3, H7
31.a.83
56.h.01
0.004
104
2
HV3-23
HV3-23
HD3-9
HD3-9
HJ6
HJ6
24
28
8%
8%
KV3-15
KV2-29
KJ2
KJ4
9
9
+++
+++
H3, H7
None
01.s.01
31.f.01
0.007
1
1
HV1-69
HV1-69
HD5-18
HD3-22
HJ4
HJ4
15
15
10%
7%
KV4-1
KV3-20
KJ3
KJ2
9
9
n.b.
+++
None
H1, H2, H5, H9
None
None
31.f.01
56.ND.11
5.1E-5
1
1
HV1-69
HV1-69
HD3-22
HD3-22
HJ4
HJ6
15
15
7%
6%
KV3-20
KV3-20
KJ2
n.r.
9
n.r.
+++
n.d.
None
n.d.
54.ND.03
56.g.01
0.001
1
1
HV1-69
HV1-69
HD3-22
HD3-22
HJ6
HJ6
13
13
8%
6%
n.d.
KV3-20
n.d.
KJ2
n.d.
9
n.d.
n.b.
n.d.
None
n.d.
None
HV7-4-1
HD3-9
HJ4
21
1%
KV3-15
KJ1
+++
H1, H5
H3
HV3-49
HD3-9
HJ4
21
9%
KV2-30
KJ1
n.d.
n.d.
n.d.
1
1
HV3-23
HV4-4
HD6-13
HD6-13
HJ4
HJ4
15
15
0%
3%
KV4-1
KV2-40
KJ1
KJ2
9
7
n.b.
n.d.
None
n.d.
None
n.d.
54.e.01
56.k.01
6.9E-5
0.001
01.i.01
56.i.01
0.032
n.a.
31.d.01
n/a
HV3-30
HD3-9
HJ3
21
4%
KV4-1
KJ2
+++
None
n.a.
01.a.44
n/a
53
HV4-34
HD2-8
HJ6
24
12%
KV1-9
KJ2
+++
H1, H2, H5
H3, H7
*IMGT CDR 3 lengths used; n.d.: not determined; : crystal structure in complex with HA determined; n.b.: no binding to HA; n.r.: not recovered; None: no pseudovirus neutralization observed; n.a.: not applicable
Figure 1. H5N1 Vaccine Recipients Have Cross-Reactive B Cells that Utilize the Same Genetic Elements and Neutralize Group 1 and Group 2
Influenza A Viruses
(A) Phylogenetic tree depicting influenza A subtypes, generated using HA sequences (one per subtype except H1, H3, H5, and H7) with program MEGA6. Scale
bar indicates distance per fractional nucleotide change.
(B) Neutralization by serum from 63 vaccinees, sampled 2 weeks after final H5N1 immunization and assessed against vaccine strain (A/Indonesia/5/2005) and
heterologous group 2 strains (H3N2: A/Hong Kong/1-4-MA21-1/1968; H7N7: A/Netherlands/219/2003). Ten subjects were selected for flow cytometric (FACS)
characterization, as highlighted in key. Dotted line indicates the limit of detection.
(C) FACS analysis of PBMC samples isolated from H5N1-vaccine recipients 2 weeks after final vaccination and co-stained with HA probes H5 (A/Indonesia/5/
2005) and H3 (A/Perth/16/2009). Sizable populations of H5-H3 cross-reactive memory B cells observed in six of ten subjects (Figure S2).
et al., 2008). In terms of reproducibility, the HV1-69-derived antibodies have the additional advantage of utilizing heavy chainonly recognition, and prior studies have shown their vaccineinduced elicitation (Khurana et al., 2013; Ledgerwood et al.,
2011, 2013; Sui et al., 2009; Wheatley et al., 2015; Whittle
et al., 2014). However, HV1-69-derived antibodies generally do
not neutralize both group 1 and group 2 strains of influenza A,
and, only a single HV1-69-derived antibody has been identified
(CR9114) capable of neutralizing both group 1 and group 2
strains of influenza A (Dreyfus et al., 2012). Other broadly neutralizing antibodies have been identified, such as FI6v3 and 39.29,
both of which derive from the HV3-30 germline gene; however,
co-crystal structures with HA reveal different modes of recognition (Corti et al., 2011; Nakamura et al., 2013), and FI6v3 and
39.29 are thus not members of the same class. Indeed, a reproducible antibody class capable of neutralizing both group 1 and
group 2 influenza A viruses has not been observed in multiple
donors.
We previously showed that subjects enrolled in the phase I
clinical trial, VRC 310who received an A/Indonesia/05/2005
monovalent inactivated virus (MIV) vaccine primed by an H5
DNA plasmid vaccine (Ledgerwood et al., 2011, 2013) (Table
S1A)showed transient expansion of H1- and H5-cross-reactive memory B cells specific to the HA stem (Wheatley et al.,
2015; Whittle et al., 2014). To determine whether these memory
B cells might encode multidonor class antibodies capable of
neutralizing group 1 and group 2 influenza A virus, we sorted
memory B cells that reacted with both H5 (group 1) and H3
(group 2) HAs. Immunoglobulin transcripts from post-vaccination cross-reactive memory B cells were sequenced, and the
encoded antibodies were synthesized and characterized. Specifically, we assessed breadth and potency of influenza A virus
neutralization, determined representative crystal structures in
complex with HA, analyzed sequence convergence based on
V(D)J-gene recombination and somatic hypermutation (SHM),
and tested sequence signatures for their ability to identify group 1
and group 2 neutralizing antibodies. Our findings reveal reproducible immunological pathways to achieve broadly reactive
antibodies and support a B cell ontogeny-based approach to obtaining a universal influenza A vaccine.
RESULTS
Identification of Memory B Cells Cross-Reactive with
Group 1 and Group 2 Influenza A HAs
We studied ten subjects from the VRC 310 H5N1 vaccine trial
who displayed a range of vaccine-elicited serum H5N1 neutralization activity, as well as varied but detectable responses against
group 2 strains A/Hong Kong/1-4-MA21-1/1968 (H3N2) or
A/Netherlands/219/2003 (H7N7) (Figures 1B and S1; Tables S1B
and S2). We used recombinant group 1-specific (H5) and group
2-specific (H3) HA probesmodified to prevent sialic acid binding
(D) Clonal diversity of H5-H3 cross-reactive B cells. The HV repertoire from each subject is shown as a pie chart; with each slice representing a unique HV clone or
clonally related family. Total number of HV sequences recovered per subject is indicated by the number at the center of each pie chart.
(E) Genetic and functional characteristics of selected antibodies recovered from H5-H3 cross-reactive B cells. Structurally characterized antibodies
indicated by .
See also Figures S1, S2, S3, and S4 and Tables S1, S2, S3, and S4.
HV6-1+HD3-3-derived antibodies were thus found in three independent donors, shared genetic elements in both the heavy
and light chain, displayed convergent affinity maturation, and
appeared to share the same mode of recognition (structurefunction analysis of recognition interface and SHM are provided
in Figure S7). Furthermore, we tested for functional complementation: swapping of heavy and light chains of the three HV61+HD3-3-derived antibodies resulted in six functional antibodies
from nine possible pairings (all three pairings with the heavy
chain of antibody 31.g.01 failed to express) (Table S7). Overall,
these results indicated the HV6-1+HD3-3-derived antibodies
form a multidonor class. Structural analysis indicated numerous
light chains to be compatible with binding, and 99% of the human population (Abecasis et al., 2012) possess alleles of the
HV6-1 and HD3-3 genes compatible with the class elicitation
and recognition described here (Figures 2D2G).
A Second Multidonor Class of Broadly Neutralizing
Antibodies with HV1-18+HD3-9 Germline Genes
Two memory B cell lineages from subjects 1 and 31 shared
immunoglobulin heavy chain sequence derived from recombination of HV1-18 with HD3-9 and HJ4 to yield highly similar amino
acid sequences in a CDR H3 of 15 amino acids (Figures 1E and
S6). Notably an Arg96HC residue was encoded by N-nucleotide
addition in both cases (Figure 3A). In each donor, the heavy chain
was paired with a light chain derived from KV2-30. Encoded immunoglobulins were expressed and shown to neutralize primarily group 1 strains of influenza A, although a few group 2 strains
were neutralized (Figure S4; Table S4). Overall neutralization
from these HV1-18+HD3-9 antibodies appeared more similar
to the group 1-specific antibody CR6261 than to the very broad
CR9114 or HV6-1-derived antibodies; nevertheless, neutralization breadth encompassed 50% of influenza A subtypes that
commonly infect humans (Figure 3B).
We determined the crystal structure of Fab 31.b.09 in complex
with A/California/04/2009 (pH1N1) HA (Figure 3C; Tables S5 and
S6). Similar to the 56.a.09-H3 complex structure, the crystallized
hemagglutinin in the Fab 31.b.09 complex was not a trimer, but a
molecular dimer (Figure S5). Despite this unexpected nontrimeric arrangement, the Ca-RMSD between the 31.b.09bound HA and the ligand-free HA in the stem region was 0.6 A,
and for clarity, we depict the 31.b.09 bound complex in a more
typical trimeric arrangement (Figure 3C). The HV1-18+HD3-9derived antibody 31.b.09 bound an epitope that overlapped
the HV6-1+HD3-3 class epitope, but with antibody rotated
105 (mostly involving a rotation perpendicular to the trimer
axis) (Figure 3C). Antibody 31.b.09 bound with both heavy and
light chains (343 A2 BSA for heavy chain and 540 A2 BSA for
the light chain). Heavy chain interactions were generated
through CDR H2 and H3 loops. In the CDR H2 (127 A2 BSA),
the HV1-18 germline-encoded Tyr53HC recognized the fusion
peptide of HA2 and Asn56HC recognized helix A of HA2, while
the CDR H3 (216 A2 BSA) was positioned over the fusion peptide-helix A interface with Ile99HC and Leu100HC inserting into
the hydrophobic groove between these two conserved elements
(Figures 3C3E). Light chain interactions involved CDR L1 and
L3, which recognized helix A (Figure 3F). We tested functional
complementation: swapping of heavy and light chains between
A
HV6-1+HD3-3
92
94
96
98
100
100b
100d
100f
102
56.a.09
footprint
Phe
100
C
CDR H3
A/Hong Kong/1-4MA21-1/1968 H3 HA0
Helix A
Met 98
Trp 21HA2
Ile 100b
56.a.09
light chain
CDR L1
Fusion
peptide
CDR H2
CDR L3
90
Thr 57
N-terminusLC
56.a.09
heavy chain
Asp 92LC
H3 HA
E
Phe
100
Tyr
50LC
Ser
53
CDR H3
Met
98
Trp 21HA2
Ile
100b
Trp
21HA2
Arg
52b
CDR L1
Tyr
33
Fusion
peptide
Helix A
Arg
50
CDR L3
CDR H2
Lys
55
Tyr
56
Asp 92 LC
Thr
57
Gln 95LC
Ile
100d
LV motif: Y at residue 33
Compatible LV genes:
30 genes compatible
Key:
= F,Y or W
x = G, A or S
= I, V, L, or M
X = any residue
HV1-18+HD3-9
92
94
96
98
100
31.b.09
footprint
A/California/04/2009
H1 HA0
CDR L1
31.b.09
light chain
CDR L3
Ile 27e
Trp 21HA2
90
Leu 100
31.b.09
CDR H3
Helix A
His 98
31.b.09
heavy chain
Fusion
peptide
CDR H2
Helix A
Trp 21HA2
Trp 21HA2
CDR H3
Helix A
Fusion
peptide
Leu
100
CDR L1
Ile 27e
CDR L3
CDR H2
Fusion
peptide
His 93
Tyr
53
His 98
Asn
56
Trp 21HA2
Trp 94
Helix A
two antibodies of the putative class resulted in functional antibodies for all four of the possible pairings (Table S7). Overall,
the results indicated the HV1-18+HD3-9-derived antibodies
614 Cell 166, 609623, July 28, 2016
HV1-18 Q-x-x-V
92
94
96
98
99
100a
100c 100d
100f
100h
100j
101
103
92
94
96
98
99
100a
100c 100d
100f
100h
100j
101
103
16.a.26
heavy chain
16.a.26
footprint
Leu 100c
16.a.26
light chain
CDR H3
NAG 38HA1
Gly 33
Val 100a
90
170
Trp 21HA2
Gln 98
Ser 52
CDR H3
Gln 98
Tyr 53
Thr 54
Gln 42HA2
16.a.26
heavy chain
Helix A
Trp 21HA2
16.g.07
footprint
16.g.07
light chain
16.g.07
heavy chain
Pro
100c
CDR H3
Val 100a
Gly 33
NAG 38HA1
90
Gln 98
170
Trp 21HA2
CDR H3
Gln 98
Tyr 53
Thr 54
Ser 52
Gln 42 HA2
16.g.07
heavy chain
Helix A
Trp 21HA2
Figure 4. A Multidonor HV1-18 Class of Broadly Neutralizing Antibodies with Q-x-x-V Motif
(A) Immunoglobulin heavy chains utilizing germline genes HV1-18, HD2-2 or HD2-15, and HJ5 or HJ2, with sequences annotated as described in Figure 2A.
(B) Neutralization breadth-potency curve for HV1-18 (Q-x-x-V) class antibodies on a panel of influenza A viruses that includes subtypes known to infect
humans.
(C) Co-crystal structure of Fab 16.a.26 (HV1-18, HD2-2, HJ5) in complex with H3-HK68. Fab heavy and light chains colored orange and lavender respectively and
depicted in surface representation while HA is depicted in ribbon and shown as a trimer. Note that labels for heavy chain residues do not explicitly show HC.
(D) The 16.a.26 CDR H3 is depicted with junction-encoded and mutated residues colored as in (A) and germline-encoded residues in orange with the antibody
footprint on the HA outlined in black.
(E) Antibody heavy chain depicted in surface representation with CDR H3 loop in ribbon. 16.a.26 Gln98HC occupies a turn in the CDR H3, which interacts with the
conserved residue Gln42HA2.
(F) Co-crystal structure of Fab 16.g.07 (HV1-18, HD2-15, and HJ2) in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) HA depicted as in (C).
(G) The 16.g.07 CDR H3 depicted as in (D) with the antibody footprint on the HA outlined in black.
92
94
96
98
A/Hong Kong/1/1968
H3 HA0
100c
100
100f
100h
31.a.83
footprint
31.a.83
footprint
31.a.83
light chain
Arg 31LC
31.a.83
light chain Ile 100
Tyr 91LC
Ile 100
70
Leu 100c
Leu 100c
Trp 21HA2
CDR H2
Met 100d
31.a.83
heavy chain
FI6v3
HV3-30
15% HV SHM
22aa CDR H3
39.29
HV3-30
10% HV SHM
18aa CDR H3
31.a.83
HV3-23
8% HV SHM
24aa CDR H3
Fusion
peptide
CDR H2
56.a.09
HV6-1
4% HV SHM
16aa CDR H3
31.b.09
HV1-18
4% HV SHM
15aa CDR H3
16.a.26
HV1-18
8% HV SHM
21aa CDR H3
16.g.07
HV1-18
11% HV SHM
21aa CDR H3
CT149
HV1-18
12% HV SHM
19aa CDR H3
CR9114
HV1-69
14% HV SHM
14aa CDR H3
Figure 5. A Conserved Site of Group 1 and Group 2 Influenza A Virus Vulnerability Targeted by Multidonor and Lineage-Unique Antibodies
(A) Neutralization breadth-potency curve for HV3-23-derived antibodies on a panel of influenza A viruses that includes subtypes known to infect humans.
(B) Immunoglobulin heavy chains utilizing germline genes HV3-23, HD3-9, and HJ6, with sequences annotated as described in Figure 2A.
(C) Co-crystal structure of Fab 31.a.83 in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) HA.
(D) Epitope for antibody 31.a.83 (outlined in black) with heavy chain depicted in yellow and junction-encoded residues (highlighted in blue), mutated residues (in
red), and germline-encoded residues (in gold) with light chain depicted in tan, HA protomer1 in gray, and HA protomer2 in dark gray. Note that labels for heavychain residues do not explicitly show HC.
(E and F) Antibodies capable of neutralizing group 1 and group 2 influenza A viruses recognize overlapping epitopes within the HA stem (colored red). The HA
surface is colored blue for structures determined previously (FI6v3 PDB: 3ZTN; 39.29 PDB: 4KVN; CT149 PDB: 4R8W; CR9114 PDB: 4FQI), and gray for
structures determined in the current study.
See also Figures S3, S4, S5, and S6 and Tables S3, S4, S5, S6, and S7.
from the same heavy chain-VDJ genes, but did not use the same
mode of recognition nor share convergent development.
Thus, antibody lineages may be multidonor (common or public), meaning that they are observed in different individuals and
share the same genetic elements and mode of recognition (Henry Dunand and Wilson, 2015; Zhou et al., 2013), or unique (uncommon or private), meaning that they have only been observed
a single time. We observed no substantial difference between
epitopes of multidonor and unique antibodies capable of neutralizing group 1 and group 2 influenza A viruses (Figures 5E and 5F),
nor did we observe segregation in antibody approach to HA used
by multidonor or unique antibodies (Figures S3E and S3F). Antibodies from multidonor lineages did, however, have lower SHM
(averaging 8% for multidonor versus 11% for the unique). The
lower SHM suggested that multidonor antibodies may undergo
more parsimonious development. We note that unique lineages
(H) The 16.g.07 CDR H3 is depicted as in (E) with Gln98HC contacting Gln42HA2.
(I) Analysis of D-gene compatibility.
(J) Analysis of V-gene compatibility.
See also Figures S3, S4, S5, S6, and S7 and Tables S3, S4, S5, S6, and S7.
Sequence
signature*
Class
PostVRC310
HeavyLight
(515,594#)
DeKosky et al.
Heavy-Light
partial
sequences
(3,019,679)
Pre-TIV
Jiang et al.
Heavy-only
(759,337)
Post-TIV
Jiang et al.
Heavy-only
(3,045,513)
Healthy
normal
donors
Heavy-only
(1,239,173)
HV6-1
+HD3-3
VH6-1 + D3-3
98MIFGI
CDR H3 = 16
21
(0.004%)
3
0
(0%)
0
0
(0%)
0
13
(0.0004%)
1
0
(0%)
0
HV1-18
+HD3-9
VH1-18
96RxxILTG
CDR H3 = 15
16
(0.003%)
3
17
(0.0006%)
1
0
(0%)
0
123
(0.004%)
3 (2)
64
(0.0052%)
2 (1)
VH1-18
53Y54T 98QxxV
CDR H3 = 17-21
309
(0.06%)
14
0
(0%)
0
0
(0%)
0 (1)
242
(0.008%)
3
2
(0.00016%)
1
HD gene family
Subject number
16
> 30 sequences
2
5 67
HV gene family
HV6-1+HD3-3
HV1-18+HD3-9
HV1-18 (Q-x-x-V)
31
10-30 sequences
HV6-1+HD3-3.1.SRP015957H+56.a.09 L
+F10 competition
HV6-1+HD3-3.2.SRP015957H+54.f.01 L
+F10 competition
HV6-1+HD3-3.2.SRP015957H+56.a.09 L
+F10 competition
54
< 10 sequences
HV1-18+HD3-9
HV1-18 Q-x-x-V
HV1-18+HD3-9.1.SRP047462H+31.b.09 L
+F10 competition
HV1-18+HD3-9.1.SRP047462
+F10 competition
HV1-18+QxxV.1. SRP047462
HV1-18+QxxV.2. SRP047462
HV1-18+QxxV.3. SRP047462
6
5
4
3
2
1
56
1
36
HV6-1+HD3-3
HV1-18+HD3-9
HV1-18 (Q-x-x-V)
HV1-18
(Q-x-x-V)
HV6-1+
HD3-3
HV1-18+
HD3-9
Control antibodies
10
0.1
0.01
0.001
0.0001
0.296 0.274 50.0 50.0
50.0 50.0
H
V6
-1
+H
H
D
V6
3-1
3.
+H
2.
H
SR
D
V1
3P0
-1
15
8+ 3.1
.S
95
H
R
D
7
P0
39.
15 H _5
1.
SR 957 4.f.
01
H
P0
H_
V1
L
56
47
-1
.a
46
8+
.0
2
H
9L
D
3- H -31
9.
.b
1.
SR .09
L
P0
47
46
2
C
R9
11
4
FI
6V
3
C
R6
26
1
C
R8
02
0
C
H6
5
Group 1 Strains
H1N1-A/South Carolina/1/1918
H1N1-A/Puerto Rico/8/1934
H1N1-A/New Caledonia/20/1999
H1N1-A/Califonia/04/2009
H2N2-A/Singapore/1/1957
H2N2-A/Canada/720/2005
H5N1-A/Vietnam/1203/2004
H5N1-A/Indonesia/05/2005
H9N2-A/Hong Kong/1073/1999
Group 2 Strains
H3N2-A/Hong Kong/1/1968
H3N2-A/Beijing/353/1989
H3N2-A/Perth/16/2009
H7N7-A/Netherlands/219/2003
H7N9-A/AnHui/1/2013
Influenza B
INF B-Brisbane/60/2008
0.2
quantify class transcripts and to identify potential class members in other subjects. Notably, despite a sequence signature
requiring residue 54HC to be altered by affinity maturation, the
HV1-18 (Q-x-x-V) class accounted for over half of the H5+ and
H3+ cross-reactive B cells we sequenced (Figure 6A) and could
be found in half the analyzed vaccinees (Figure 6B).
For other subjects, we searched published human antibody
datasets, both those with paired heavy-light sequences
(DeKosky et al., 2015), as well as those with heavy chain-only sequences (Figure 6A; Table S1C). Searches with the HV61+HD3-3 signature did not yield sequence matches in paired
heavy-light sequences, but in the published heavy chain-only
datasets, we found 13 matches, which appeared to derive
from a single lineage (Figure S6). We chose both consensus as
well as the sequence closest to consensus to synthesize and
reconstitute with light chains of HV6-1+HD3-3 class antibodies
from subjects 54 and 56. One of these reconstituted antibodies
did not express, but the other three did and bound H1 HA in a
manner that could be competed with antibody F10 (Figure 6C).
We tested two of these antibodies and both neutralized group
1 and group 2 influenza A strains (Figure 6D; Table S3), and
the neutralization signatures (Georgiev et al., 2013) of the synthesized antibodies clustered in a dendrogram with known HV61+HD3-3 class antibodies (Figure 6E).
With the HV1-18+HD3-9 signature, we found 17 matches in
published paired heavy-light chain sequences, which appeared
to derive from a single lineage (Figure 6A). We synthesized
consensus sequences and reconstituted published heavy and
light chains as well as the published heavy chain and light chain
of this class from subject 31. Both of these reconstituted antibodies bound H1 hemagglutinin in a manner that could be
competed with antibody F10 (Figure 6C), neutralized group 1
influenza A strains (Figure 6D; Table S4), and clustered in a
neutralization dendrogram with known HV1-18+HD3-9 class
antibodies (Figure 6E). The neutralization breadth of these antibodies was lower than those isolated from VRC 310 subjects,
likely due to the use of germline sequences to complete the
CDR L1 and CDR L2 regions of this antibody (the somatic mutation of 27ELC to Ile is required for optimal recognition)
(Figure 3F).
With the HV1-18 (Q-x-x-V) signature, searches did not yield
sequence matches in paired heavy-light sequences, but in
published heavy chain-only datasets (Table S1C), we found
244 matches, which appeared to derive from 4 lineages (Figures 6A and S6). We synthesized four sequences (consensus
or closest NGS read) and reconstituted with the five light
chains used previously in swapping experiments (Table S7).
None of these reconstituted antibodies bound a set of HAs
(Figure 6C) or neutralized any of the 15 viruses in our neutralization panel. Analysis of the tested heavy chains indicated that
their CDR H3 length matched that of CT149 in three of four
cases, but was below the 78% identity threshold that correlated with function in heavy-light complementation of this class
(Table S7).
Altogether, the results indicate sequence signatures with sufficient specificity to identify other functional class members by
sequence alone could be obtained for two multidonor classes:
HV6-1+HD3-3 and HV1-18+HD3-9. The sequence signature
for the third class, HV1-18 (Q-x-x-V), was complicated by incompatibility of some heavy-light pairs from this class; nonetheless,
sequence searches for this third class did place an upper limit on
the prevalence of this multidonor antibody class in the searched
databases.
Vaccine Induction of Multidonor Broadly Neutralizing
Antibodies
In the VRC 310 trial, we observed a significant expansion
(p = 0.0284) of H5+H3+ memory B cells following H5 DNA
prime-MIV boost, ranging from an increase of 1.2- to 10.6-fold
(Figure 7A). Notably, subjects with the largest increases and
the highest frequencies of multidonor antibodies had the largest
percentages of antibodies belonging to the three multidonor
classes identified here (Figure 7B). Our initial observation of a
high number of transcripts with multiple genetic commonalities
may be explained by the preferential expansion of multidonor
class transcripts; indeed, the fold-increase in cross-reactive B
cells by subject correlated with the percentage of antibody sequences with inter-subject genetic commonalities (Figure 7C).
Importantly, the frequency of cross-reactive memory B cells
post-VRC 310 vaccination correlated with the sequence signature-identified prevalence of multidonor class antibodies
(p = 0.0045) (Figure S6D). Moreover, while we did not see significant correlation between fold increase in overall sera titer versus
an increase in cross-reactive memory B cells, we did observe a
significant correlation in titers with the H1N1 virus, A/Singapore/
8/1986, which we previously found to be especially sensitive to
neutralization by stem-directed antibodies (Lingwood et al.,
2012) (Figure 7D).
To quantify the frequency of multidonor class antibodies in
unvaccinated subjects, we examined NGS-determined memory
B cell transcripts from healthy normal donors (Figure 6A). To
compare frequencies from VRC 310-vaccinated versus unvaccinated subjects, we equated the total number of sorted memory
B cells with the total number of transcripts and observed a substantially higher transcript frequency (and to a lesser degree,
a higher lineage frequency) for multidonor class sequences on
H5N1 vaccination in the VRC 310 trial (Figure 7E).
We also examined published antibody sequences from subjects immunized with the 2009 or 2010 seasonal influenza
vaccine (Jiang et al., 2013) (Figures 6A, 7F, and S6). Overall, transcripts matching the HV1-18+HD3-9 signature were 103 more
prevalent prior to vaccination than the other multidonor transcripts; however, this class appeared not to expand on either
seasonal or VRC 310 vaccination. By contrast, transcripts
matching HV6-1+HD3-3 and HV1-18 (Q-x-x-V) signatures appeared to be present at low frequency prior to vaccination, to increase on seasonal vaccination, and to increase up to 1,000-fold
on VRC 310 vaccination (Figure 7F).
DISCUSSION
The vaccine induction of broadly neutralizing antibodies against
influenza A virus has been a long standing immunological goal.
While the human immune system can generate antibodies
capable of neutralizing group 1 and group 2 strains of influenza
(Corti et al., 2011; Dreyfus et al., 2012; Nakamura et al., 2013;
Cell 166, 609623, July 28, 2016 619
Figure 7. Vaccine Induction of Antibodies Capable of Neutralizing Group 1 and Group 2 Influenza A Viruses
(A) Frequencies of H5-H3 cross-reactive memory B cells pre- and post-H5N1 vaccination. Subjects for whom pre-immunization samples were no longer available
are indicated with open symbols; subject name and fold increase shown for others.
(B) Frequency of multidonor class sequences by donor and multidonor class.
(C) Fold increase in cross-reactive B cells relative to prevalence of heavy chain sequences with three (out of a possible four) of the same heavy chain genetic
elements in at least one sequence in any of the six analyzed subjects (Pearson correlation with the total number of sequences provided in Figure 1D).
(D) Fold increase in cross-reactive B cells relative to the fold increase in sera neutralization titer for all tested influenza A strains (shown in black) (see Figures 1 and
S1), or the single H1N1-A/Singapore/8/1986 strain (shown in red).
(E) Bar graphs of transcript frequencies (left) and lineage frequencies (right). Left: frequency of multidonor-class transcripts by dataset. Right: frequency of
multidonor class lineages for each dataset.
(F) Transcript frequency versus dataset and goal. Stars depict upper-bound estimates, and circles depict frequencies confirmed by neutralization.
(G and H) Multidonor antibodies displayed in ribbon with class-conserved contact residues shown in stick. Antibody epitopes shown in purple (HV6-1+HD3-3),
green (HV1-18+HD3-9), and orange (HV1-18 with Q-x-x-V) with black outlines. Glycans shown in surface representation and colored by conservation: conserved
(light green) or variable (dark green).
See also Figures S3 and S6 and Tables S3 and S4.
1,000-fold increases in the transcript frequencies for two multidonor classes of influenza A neutralizing antibodies could be
achieved through immunization with a divergent influenza (Figures 6A and 7F), likely by enhancing immune focus to the HA
stem (Figures 7G and 7H), an approach utilized by stem-only
or headless immunogens (Impagliazzo et al., 2015; Yassine
et al., 2015) and by chimeric HA immunogens (Krammer et al.,
2015). It will be fascinating to evaluate how these immunogens,
in various vectored, subunit, and prime-boost combinations, will
fare at further inducing, maintaining, or expanding the multidonor
broadly neutralizing antibodies identified here.
EXPERIMENTAL PROCEDURES
Ethics Statement and VRC 310 Study Design
The VRC 310 study protocol and associated procedures were approved by the
National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board. All participants provided written informed consent in accordance
with the Declaration of Helsinki. The VRC 310 study (ClinicalTrials.gov identifier
NCT01086657) (Ledgerwood et al., 2011, 2013) was conducted by the Vaccine
Research Center, NIH. See Table S1 and the Supplemental Experimental Procedures for details.
Expression of HA Probes, Flow Cytometry, Cell Sorting, and
Sequencing
HA constructs consisting of the extracellular domain of HA modified to ablate
sialic acid binding and C-terminally fused to (1) a T4-fibritin trimerization motif,
(2) a biotinylatable AviTag sequence, and (3) a hexahistidine affinity tag were
synthesized (Genscript), cloned into a CMV-expression plasmid, and expressed as previously described (Whittle et al., 2014). Cryopreserved PBMC
samples were stained and sorted on a fluorescence-activated cell sorting
(FACS) Aria II using fluorescently labeled recombinant H1 (A/New Caledonia/
20/1999), H5 (A/Indonesia/05/2005), or H3 (A/Perth/16/2009) probes; single
memory B cells binding to both H5 and H3 probes were sorted, and
sequencing of immunoglobulin genes by multiplex PCR was performed as previously described (Whittle et al., 2014). See the Supplemental Experimental
Procedures for details.
Production of Pseudotyped Lentiviral Vectors and Influenza A
Viruses and Measurement of Antibody Neutralizing Activity
Influenza HA pseudotyped lentiviral vectors expressing a luciferase reporter
gene were produced and used to infect 293A cells. All influenza viruses
used in the microneutralization assays were expanded in Madin-Darby canine
kidney epithelial (MDCK) cells in the presence of Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) and titrated in MDCK cells.
See the Supplemental Experimental Procedures for details.
Structural Analysis and Sequence Bioinformatics
Both negative stain-EM and X-ray crystallography were used to characterize
antibodies from the VRC 310 trial and their complexes with HA. An Antibodyomics1.0 pipeline, modified to analyze both 454 and Illumina output,
was used to analyze B cell transcripts for the presence of sequence signatures specific to multidonor antibodies. Frequentist probabilities were used
to determine likelihoods of sequence convergence and germline prevalence
in the human population. See the Supplemental Experimental Procedures for
details.
ACCESSION NUMBERS
The accession numbers for the coordinates and structure factors reported in
this paper are Protein Data Bank (PDB): 5K9J, 5K9K, 5K9O, 5K9Q, 5KAN,
and 5KAQ. The accession numbers for the sequences for all of the antibodies
reported in this paper are GenBank: KX386124KX387227. The accession
numbers for the NGS data reported in this paper are Short Reads Archive:
SRP026397 and SRP073039.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.06.043.
AUTHOR CONTRIBUTIONS
M.G.J., A.K.W., P.V.T., G.-Y.C., C.S., P.D.K., J.R.M., and A.B.M. conceived,
designed, and coordinated the study. M.G.J., A.K.W., P.V.T., G.-Y.C., C.S.,
L.S., P.D.K., J.R.M., and A.B.M wrote and revised the manuscript and figures.
B.S.G. and J.E.L. carried out VRC 310 trial and provided subject samples.
R.A.K. and M.R. provided support for B cell analysis and sorting. J.C.B.,
M.K., and J.R.W. designed HA constructs. M.G.J., A.K.W., P.V.T., R.T.B.,
A.D., R.A.G., M.K., W.-P.K., K.L., S.N.N., M.S.P., E.S.Y., B.Z., Y.Z., M.A.,
S.D., C.R.L., A.R., L.W., X.W., H.M.Y., C.S., Y.M., Y.T., U.B., and NISC CSP
performed experiments. M.G.J., G.Y.-C., C.S., C.-H.S., and P.D.K. carried
out bioinformatics analysis. M.G.J., A.K.W., P.V.T., G.-Y.C., C.S., R.T.B.,
I.S.G., M.K., W.-P.K., K.L., T.B., S.D., Y.T., U.B., J.C.M., K.S., D.C.D., L.S.,
P.D.K., J.R.M., and A.B.M. analyzed data. All authors read and approved the
manuscript. Detailed author contributions are provided in Supplemental
Information.
ACKNOWLEDGMENTS
We thank D. Ambrosak and R. Nguyen for assistance with flow cytometry;
J. Chrzas, J. Gonczy, U. Chinte, and staff at Southeast Regional Collaborative Access Team (SER-CAT) for help with X-ray diffraction data collection;
G. Georgiou and S.R. Quake for human antibody sequences; J. Stuckey
for assistance with figures; and members of the Structural Biology Section,
Structural Bioinformatics Core Section and Virology Laboratory of the Vaccine Research Center for helpful comments. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center
and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH. This work was supported in part with federal funds
from the Frederick National Laboratory for Cancer Research, NIH, under
contract HHSN261200800001E. Use of insertion device 22 (SER-CAT) at
the Advanced Photon Source was supported by the U.S. Department of
Energy, Basic Energy Sciences, Office of Science, under contract W-31109-Eng-38.
Received: January 22, 2016
Revised: April 12, 2016
Accepted: June 23, 2016
Published: July 21, 2016
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Article
Authors
Andrea J. Wolf, Christopher N. Reyes,
Wenbin Liang, ..., K. Mark Coggeshall,
Moshe Arditi, David M. Underhill
Correspondence
david.underhill@csmc.edu
In Brief
The metabolic enzyme hexokinase
unexpectedly acts as a pattern
recognition receptor that recognizes
bacterial peptidoglycan and triggers
activation of inflammasomes.
Highlights
d
Article
Hexokinase Is an Innate Immune Receptor for the
Detection of Bacterial Peptidoglycan
Andrea J. Wolf,1,2 Christopher N. Reyes,1 Wenbin Liang,3,6 Courtney Becker,1 Kenichi Shimada,2,4 Matthew L. Wheeler,1,2
Hee Cheol Cho,3,7 Narcis I. Popescu,5 K. Mark Coggeshall,5 Moshe Arditi,2,4 and David M. Underhill1,2,*
1F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles,
CA 90048, USA
2Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
3Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
4Division of Pediatric Infectious Diseases, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
5Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
6Present address: University of Ottawa Heart Institute and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
ON K1Y 4W7, Canada
7Present address: Departments of Biomedical Engineering and Pediatrics, Emory University, Atlanta, GA 30322, USA
*Correspondence: david.underhill@csmc.edu
http://dx.doi.org/10.1016/j.cell.2016.05.076
SUMMARY
found that degradation of Staphylococcus aureus in phagosomes is a key factor in determining the types and amounts
of inflammatory cytokines produced following phagocytosis (Ip
et al., 2010; Wolf et al., 2011; Muller et al., 2015). In particular,
we noted that production of interleukin (IL)-1b and IL-18 required
the degradation of S. aureus cell wall peptidoglycan (PGN) and
that this response is suppressed when the organism modifies
its PGN to become resistant to degradation (Shimada et al.,
2010).
IL-1b and IL-18 play essential roles in controlling bacterial infections, in part, by recruiting neutrophils to sites of infection
and polarizing T cell responses. Unlike many other cytokines,
IL-1b and IL-18 are transcribed as pro-cytokines in the cytosol.
Signaling to multiprotein complexes known as inflammasomes
activates caspase-1 to process and secrete the cytokines (Lamkanfi and Dixit, 2014; Martinon et al., 2004). While there are
several varieties of inflammasomes, the one responsible for responding to PGN is defined by the presence of NOD-like receptor family, pyrin domain-containing 3 (NLRP3). The mechanism
by which NLRP3 is activated by PGN is not known. It is generally
thought that all of the immunomodulatory activity of the S. aureus
PGN comes from the degradative release of muramyl dipeptide
(MDP), which is detected by cytosolic NOD2 receptor. However,
we observed that NLRP3 inflammasome activation in response
to S. aureus PGN was not affected by the loss of NOD2 (Shimada
et al., 2010). Thus, the fragment of PGN that must be generated
through degradation to activate the inflammasome and how it is
sensed have not been established.
Diverse particulate stimuli that activate the NLRP3 inflammasome have been identified, including crystals such as silica,
alum, asbestos, uric acid, and cholesterol (Dostert et al., 2008;
Duewell et al., 2010; Hornung et al., 2008; Martinon et al.,
2006). Like PGN particles, phagocytosis of these crystals is a
necessary step in the process leading to inflammasome activation. For crystalline particles, which are non-degradable and
non-microbial, it has been suggested that disruption of the
phagosomal compartment leads to NLRP3 inflammasome activation (Hornung et al., 2008). However, we have previously
observed that phagosomes containing PGN remain intact, and
624 Cell 166, 624636, July 28, 2016 2016 Published by Elsevier Inc.
Nlrp3-/-
0.8
3.0
0.6
2.0
0.5
0.7
2.5
Sup
***
0.3
***
0.2
dT
0.1
SA
PGN
pd
A:
U
T
AT
P
0.5
Strep
PGN
IL-1 p45
Lys
***
***
Tubulin
BS
PGN
D
UT
PGN
ATP
Nig
IL-1 (ng/ml)
4
3
2
1
0
5
34
64
77
K+ (mM)
92
121 150
S. aureus
ATP
16
14
12
10
8
6
4
2
0
E
UT
% LDH release
C
6
IL-1 p17
***
0.4
1.5
1.0
IL-1 (ng/ml)
WT
0.9
3.5
IL-1 (ng/ml)
***
1.0
4.0
T
AT
P
SA
St PG
re N
p
BS -PG
-P N
G
N
64 92 150
K+ (mM)
45
40
35
30
25
20
15
10
5
0
ATP
20
40
Time (h)
PGN
60
80
Figure 1. PGN Activates the NLRP3 Inflammasome Independent of Potassium Efflux and Cell Death
(A) LPS-primed BMDMs from wild-type and Nlrp3 / mice were untreated (UT) or stimulated with 5 mM ATP for 2 hr or pdA:dT or PGN from DoatA S. aureus (SA),
Streptomyces (Strep), or B. subtilis (BS) at 2040 mg/ml, and IL-1b was assayed in the supernatant after 6 hr.
(B) Immunoblot of mature IL-1b in supernatants (Sup) or pro-IL-1b in cell lysates (Lys) of wild-type macrophages stimulated as in (A).
(C and D) LPS-primed BMDMs were stimulated in the presence of increasing concentrations of extracellular KCl with (C) 20 mg/ml PGN 6 hr, 5 mM ATP 2 hr,
10 mg/ml nigericin (Nig) 2 hr, or (D), S. aureus (DoatA) 6 hr.
(E) LPS-primed BMDMs were stimulated with PGN (20 mg/ml) or ATP (5 mM), and release of lactate dehydrogenase (LDH) into supernatants was measured at
indicated times and shown as percentage of maximum at each time point.
Error bars indicate SD. ***p < 0.001.
See also Figure S1.
process that, as we have previously shown, requires degradation (Shimada et al., 2010). The diminished IL-1b production by
Nlrp3 / BMDMs in response to PGN are not a consequence
of differential phagocytosis or lysosomal enzyme activity, which
are equivalent in wild-type and Nlrp3 / BMDMs (Figures S1A
S1C). In the process of evaluating inflammasome activation in
response to PGN, we observed some behaviors inconsistent
with current models of mechanisms of activation. First, it has
been suggested that efflux of cytosolic potassium is essential
for NLRP3 inflammasome activation (Munoz-Planillo et al.,
2013). While we confirmed that IL-1b secretion triggered by
ATP or nigericin in lipopolysaccharide (LPS)-primed macrophages is strongly inhibited by extracellular potassium, we found
that PGN-induced IL-1b secretion is not affected by extracellular
potassium (Figure 1C). We also observed no effect of extracellular potassium on IL-1b secretion in response to whole DoatA
S. aureus (Figure 1D), a strain that makes a PGN that is
highly sensitive to phagosomal degradation and that we have
previously shown to be a strong activator of the NLRP3 inflammasome (Shimada et al., 2010). Second, unlike many inflammasome activators, PGN-induced caspase-1 activation does not
result in pyroptosis, as measured by the release of lactate dehydrogenase (LDH) (Figure 1E), annexin V staining (Figure S1D),
or propidium iodide uptake (Figure S1E). We only observe
background levels of cell death over 3 days in macrophages
stimulated with PGN (Figure 1E). Compared to classic NLRP3
activators like ATP and nigericin, PGN-induced inflammasome
activation occurs over a longer time period; for example, in Figure 1A, PGN induces much less IL-1b over 6 hr than ATP triggers
in 2 hr. Given these unique features of PGN-induced activation,
we set out to determine how PGN is sensed by macrophages.
NAG Is the Minimal Inflammasome-Activating
Component of PGN
PGN, which makes up as much as 80% of the dry weight of typical
Gram-positive bacteria, is a polysaccharide of repeating units of
N-acetylmuramic acid (NAM; MurNAc) and NAG (GlcNAc) crosslinked by short amino acid side chains (Figure 2A).
MDP, the NOD2-activating fragment of PGN, has been
suggested to stimulate IL-1b release under certain conditions
(Faustin et al., 2007; Ferwerda et al., 2008; Hsu et al., 2008;
Marina-Garca et al., 2008; Martinon et al., 2004; Pan et al.,
2007). However, when we treated macrophages with soluble
MDP or lipofectamine-complexed MDP (to deliver it to the
cytosol), they did not mimic the response to PGN that we saw
(Figure 2B). Furthermore, as noted earlier, we have previously
shown that PGN-induced IL-1b secretion is not blocked in macrophages lacking NOD2 (Shimada et al., 2010). Together, the
data suggest that some other lysosomal degradation product
of PGN must be detected.
Therefore, we examined the inflammasome-activating capacity of other potential PGN degradation products. We observed
that the NAG sugar subunit from the backbone of PGN becomes
a potent activator of IL-1b processing and secretion in LPSprimed macrophages when it is complexed with lipofectamine
to deliver it to the cytosol (Figure 2C). We found that delivery
of NAG to the cytosol is important to its function, since soluble NAG only induced IL-1b when added to culture medium
626 Cell 166, 624636, July 28, 2016
B
NAM
IL-1 (ng/ml)
L-Ala
D-Ala
D--Glu
L-Lys
(Gly)5
L-Lys
D--Glu
D-Ala
L-Ala
NAG
NAM
D
1.4
2.0
1.2
NAG
IL-1 (ng/ml)
NAG
C
2.5
1.5
1.0
NAG
U
T
AT
P
Li
po
LN
AG
1.0
IL-1 p45
Lys
0.8
Tubulin
0.6
0.4
0.5
IL-1 p17
Sup
0.2
U
T
Li
sM po
L- DP
M
pd DP
A:
dT
0
Lipo
L-NAG
Blue = DAPI
po
LN
L- AG
G
A
L- M
G
L- lc
Su
c
M
DP
PG
N
LP
S
Li
L-
L-
Li
L-
G
A
L- M
G
L- lc
Su
c
AM
100
po
AG
0.3
0.2
0.1
po
200
0.4
Li
300
dT
400
0.5
A:
500
10
pd
AT
P
600
12
WT
700
TNF (ng/ml)
800
IL-1 (ng/ml)
IL-1 (pg/ml)
L-NAG
Lipo
L-NAG
10
dT
2 6 8 20 2 6 8 20 2 6 8 20 2 6 8 20 h
L-
K+ (mM)
A:
34 64 77 92 121 150
pd
0
5
AG
20
0.2
po
0.4
30
Li
0.6
40
0.8
50
1.0
LDH % of Max
IL-1 (ng/ml)
60
L-NAG
1.2
(G) Unprimed BMDMs were treated with lipofectamine-complexed sugars as in (F), and TNF-a was measured in the supernatant after 6 hr.
(H) LPS-primed BMDMs from wild-type (WT) and Nlrp3 / mice were stimulated as described above.
(I) LPS-primed BMDMs were stimulated with lipofectamine-complexed NAG in the presence of increasing concentrations of extracellular KCl for 6 hr.
(J) LPS-primed BMDMs, stimulated as described above, were assessed for LDH release at indicated times.
Error bars indicate SD. ***p < 0.001, Students t test.
See also Figure S2.
20
18
16
14
12
10
8
6
4
2
0
- + + + + + LPS
1 2 4 h
PGN
D
14
120
12
100
NAG
NAM
Suc
human
HK Activity (U/ml)
mouse
HK Activity (U/ml)
**
80
60
40
20
25
50
75
Concentration (mM)
100
10
8
6
4
2
UT
MDP
AT
P
Cytosolic mtDNA
Fold Induction
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
% Actiivity
T
U
TM
PGN
1
L-NAG
3
hr
Tubulin
Tom20
Tom20
CytoC
Antibody
Specificity
Ctl
U
T
PG
N
Li
po
LN
AG
HK activity
(% of Total Cell Activity)
Before
*
Antibody
Specificity
Ctl
cytosol
NAG
***
Tubulin
I
3h
hr
HK2
H
20
18
16
14
12
10
8
6
4
2
0
HK2
CytoC
cytosol
Lipo
HK2 (ng/ml)
0.1 1 5
NAG (mM)
**
5
***
4
3
***
**
2
1
0
1 2 3 1 2 3 1 2 3 1 2 3 h
UT
PGN L-NAG pdA:dT
After
HK2
GFP
Mito
DsRed
Before
NAM
After
HK2
GFP
Mito
DsRed
Figure 4. Hexokinase Is the Receptor that Detects NAG for Inflammasome Activation
(A) LPS-primed BMDMs were stimulated with ATP (5 mM) for 2 hr or PGN (40 mg/ml) as indicated, and the presence of mtDNA in the cytosolic fraction was
measured by RT-PCR.
(B and C) Hexokinase (HK) activity in purified mouse macrophage mitochondria was assessed in the presence of increasing concentrations of (B) NAG, NAM,
sucrose, or (C) MDP (25 mM). UT, untreated.
(D) NAG inhibits the activity of purified human hexokinase.
(E and F) Hexokinase in the cytosol fraction of LPS-primed BMDMs stimulated for the indicated times with PGN (40 mg/ml, from S. aureus) or lipofectaminecomplexed NAG was detected by immunoblot. Clotrimazole (CTM) treatment was used as a positive control for hexokinase release from mitochondria, and
mitochondrial markers Tom20 and cytochrome c (CytoC) were included to control for mitochondrial integrity. Control lysates were included (Antibody Specificity
Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). Lipo, lipofectamine.
(G) LPS-primed BMDMs were stimulated as indicated PGN (40 mg/ml), and hexokinase 2 in the cytosol was determined by ELISA.
(H) LPS-primed BMDMs were stimulated as indicated, and cytosolic hexokinase enzyme activity was measured.
(I) LPS-primed BMDMs expressing hexokinase 2 fused to GFP (HK2-GFP) and DsRed targeted to mitochondria (Mito-DsRed) were microinjected with NAG or
NAM as indicated. Association of hexokinase with mitochondria was visualized before and 1 min after injection (n = 10 cells assessed for each sugar).
Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Students t test.
See also Figure S4.
Figure 5. Hexokinase Dissociation from Mitochondria Is Sufficient to Activate the NLRP3 Inflammasome
(A) LPS-primed BMDMs were treated with cell-permeable hexokinase dissociation peptide (HKVBD) or scrambled control peptides (Ctl) fused to TAT peptide
(20 mM) for the indicated times, and the amount of hexokinase in the cytosolic fraction was determined by immunoblot. Control lysate was included on each gel
(Antibody Specificy Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). UT, untreated.
(B) LPS-primed BMDMs expressing HK2-GFP and mitochondria-localized DsRed (Mito-DsRed) were imaged following treatment with HKVBD and Ctl peptides
fused to TAT (20 mM) to assess hexokinase redistribution.
(C and D) LPS-primed BMDMs were treated with HKVBD or control peptides fused to cell-permeable antennapedia peptide; IL-1b (C) and IL-18 (D) were
measured by ELISA after 2 hr.
(E) LPS-primed BMDMs from wild-type and Nlrp3 / mice were stimulated with 5 mM ATP, pdA:dT, HKVBD or control peptides fused to antennapedia peptide,
and IL-1b was measured in the supernatant after 2 hr.
(F and G) LPS-primed BMDMs were treated with HKVBD or control peptides fused to TAT peptide; (F) cleaved IL-1b and caspase-1 were detected by immunoblot at
2 hr, and (G) inflammasome assembly was observed by NLRP3 and caspase-1 p10 proximity ligation (PLA, red). Nuclei were stained with DAPI (blue). Sup, supernatant.
High levels of G6P trigger the release of hexokinase from mitochondria and, thus, reduce the rate of further G6P production
(Gerber et al., 1974; Pastorino et al., 2002). Therefore, we predicted that excess G6P would activate the NLRP3 inflammasome. Indeed, when we treated primed BMDMs and bone
marrow-derived dendritic cells (BMDCs) (data not shown) with
lipofectamine-complexed G6P, we observed IL-1b secretion
by ELISA (Figure 6A) and production of cleaved IL-1b p17 and
caspase-1 p10 in the supernatant (Figure 6B). This activation
was NLRP3 dependent (Figure S6A). Consistent with its role as
a hexokinase inhibitor, we observed increased hexokinase in
the cytosol following G6P treatment (Figure 6C). 2-deoxyglucose
(2-DG) is a glycolytic inhibitor that is commonly used in studies
of cell metabolism. It competes with glucose in the glycolytic
pathway. When we treated primed BMDMs with 2-DG, we
observed dose-dependent induction of IL-1b by ELISA (Figure 6D), as well as cleaved IL-1b p17 and caspase-1 p10 in the
supernatant (Figure 6E), as has recently been reported by others
(Nomura et al., 2015). However, 2-DG does not function as an
inhibitor of hexokinase like NAG or G6P. Instead, 2-DG is metabolized by hexokinase to 2-deoxyglucose-6-phosphate (2-DG6P)
(Figure S4C), which cannot be utilized by downstream glycolytic
enzymes (Wick et al., 1957). The result is a buildup of 2-DG6P
that inhibits hexokinase like G6P but is sensitive to the presence
of glucose, hexokinases preferred substrate. Thus, while 2-DG
can trigger inflammasome activation in primed macrophages in
media containing glucose, it is more effective in the absence of
glucose (Figure 6F) and is completely dependent on NLRP3 (Figure S6B). Consistent with our previous observations, 2-DG treatment leads to an increase in hexokinase in the cytosol of BMDMs
(Figure 6G). Lastly, we treated primed cells with citrate, a natural
intermediate in the tricarboxylic acid (TCA) pathway that inhibits
phosphofructokinase when it accumulates. Buildup of citrate
thus backs up the glycolytic pathway and naturally elevates
cytosolic G6P levels (Berg et al., 2002). As expected, treating
primed cells with citrate triggered NLRP3-dependent IL-1b
production and caspase-1 cleavage (Figures 6H and S6C). While
these metabolic stresses can cause cell death under certain
conditions, we observed IL-1b production under conditions
that do not cause substantial cell death (Figure S6D). These
data suggest an intriguing relationship between cellular metabolism and inflammatory signaling.
DISCUSSION
While the original evolutionary role of phagocytosis was to eat
and degrade other microbes to obtain nutrients, this study
suggests that mammalian phagocytes have adapted the cells
metabolic machinery for utilizing these nutrients to detect the
presence of microbial-derived sugars and metabolic perturbation as danger signals. In this study, we have shown that the
NAG subunit of the sugar backbone of bacterial PGN induces
inflammasome activation by inhibiting hexokinase, the first
(H and I) Indicated mice were injected i.p. with 500 ml of PBS (n = 6), 240 mM HKVBD (n = 7), or control peptide fused to TAT peptide (n = 6); peritoneal cavities were
lavaged after 4 hr; total cells were counted; and neutrophil content was determined by flow cytometry (both experiments were each done 13). ns, not significant.
Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Students t test (CE and H) and one-way ANOVA and Newman-Keuls multiple comparison test (G).
See also Figure S5.
H
G
step in glycolysis. The inhibition of hexokinase results in hexokinase dissociation from the mitochondria, which, as we have
observed, is sufficient to initiate an NLRP3 inflammasomeactivating cascade in the cell. This model is supported by the
observation that several metabolic perturbations that inhibit
hexokinase function, such as treatment with glucose-6-phosphate, 2-deoxyglucose, or citrate, all lead to inflammasome
activation. How exactly hexokinase release from the mitochondrial outer membrane promotes NLRP3 inflammasome activation remains to be understood.
Mitochondrial dynamics have been broadly implicated in regulation of the NLRP3 inflammasome, including studies demonstrating a role for mitochondrial movement along microtubules
(Misawa et al., 2013), regulation of mitochondrial fission and
growth (Park et al., 2015), mitophagy (Zhong et al., 2016), and
release of mtDNA into the cytosol (Shimada et al., 2012; Zhong
et al., 2016) in modulating inflammasome activation. NAG inhibition of hexokinase was particularly interesting, because hexokinases I and II, the primary isoforms that regulate glycolysis, are
known to associate with the VDAC in the mitochondrial outer
membrane (John et al., 2011; Pastorino and Hoek, 2008; Pastorino et al., 2002; Rasola et al., 2010). The VDAC is known to regulate mitochondrial ROS (reactive oxygen species) production
(da-Silva et al., 2004), is a suggested component of the mitochondrial permeability transition pore that can release large molecules (including mtDNA) into the cytosol (Rasola et al., 2010;
Tomasello et al., 2009), and is localized to regions enriched for
cardiolipin (Sun et al., 2012) an NLRP3 activator. The interaction
of hexokinase with the VDAC on the outer membrane of mitochondria provides hexokinase with preferential access to newly
produced ATP transported from the matrix by the VDAC (Pastorino and Hoek, 2008). Hexokinase inhibition and dissociation from
the mitochondria constitute an essential step in regulation of the
rate of glycolysis (da-Silva et al., 2004; Pastorino and Hoek,
2008). Excess glucose-6-phosphate generated by hexokinase
leads to feedback inhibition of hexokinase and its dissociation
from mitochondria, slowing glycolysis. In addition, the interaction of hexokinase with the VDAC protects cells from mitochondrial ROS production (da-Silva et al., 2004) and suppresses
pro-apoptotic interactions between the VDAC and Bcl-family
members (Bax, Bid, etc.), which promotes sustained opening
of the mitochondrial permeability transition pore (Chiara et al.,
2008; Majewski et al., 2004; Pastorino and Hoek, 2008; Pastorino et al., 2002; Rasola et al., 2010). Each of these processes
have been previously implicated in NLRP3 inflammasome regulation, but how they relate to microbial sensing has not been
understood (Shimada et al., 2012; Zhou et al., 2011).
At first thought, NAG would seem to be a poor candidate to be
a PAMP detected by the innate immune system, since it is not
unique to bacteria. However, free NAG is not generally found in
the cytosol of mammalian cells and is primarily generated only
in small amounts following degradation of glycosylated proteins.
In biosynthetic pathways, uridine diphosphate (UDP)-NAG is
synthesized directly from glycolytic intermediates and utilized
in glycosylation processes without existing as free NAG. In
contrast, during degradation of particulate PGN in phagosomes,
unusually large amounts of NAG can be expected to become
available. The presence of a transporter that moves NAG from
634 Cell 166, 624636, July 28, 2016
for 1 hr and washed. Cells were either imaged or treated with PBS + proteinase
K (1 U/ml) to remove unbound particles and analyzed by flow cytometry to
determine the degree of phagocytosis.
support came from the Janis and William Wetsman Family Chair in Inflammatory Bowel Disease at Cedars-Sinai Medical Center (D.M.U.). Thanks to the
laboratory of Dr. Robin Shaw for use of their FemtoJet microinjection system.
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Article
Authors
Elvan Boke, Martine Ruer, Martin Wuhr, ...,
David Drechsel, Anthony A. Hyman,
Timothy J. Mitchison
Correspondence
elvan_boke@hms.harvard.edu
In Brief
Amyloid-like self-assembly of a specific
protein drives formation of a cellular
compartment in oocytes.
Highlights
d
Article
Amyloid-like Self-Assembly
of a Cellular Compartment
Elvan Boke,1,* Martine Ruer,2 Martin Wuhr,1,3 Margaret Coughlin,1 Regis Lemaitre,2 Steven P. Gygi,3 Simon Alberti,2
David Drechsel,2 Anthony A. Hyman,2 and Timothy J. Mitchison1
1Department
SUMMARY
B
2 M NaCl
Balbiani
body
50 m
Bb
95C
N
50 m
Time (min)
10 m
2 m
0.5 m
50 m
high salt (2 M NaCl) (Figure 1B, first panel) and high temperature
(up to 95 C) (Figure 1B, second panel). Confirming previous work
on intact oocytes, thin-section electron microscopy of the isolated Balbiani bodies revealed densely packed mitochondria,
ER, and Golgi stacks as well as compact fibrillar elements that
others have shown to be made of ribonucleoprotein (RNP) (Figure 1C) (al-Mukhtar and Webb, 1971; Balinsky and Devis,
1963; Billett and Adam, 1976).
To probe the molecular properties of the Balbiani body, we
introduced a number of small molecules into the oocytes. Thioflavin T (ThT), a dye that stains the b sheet-rich structures of amyloid (Alberti et al., 2010; Nilsson, 2004), strongly accumulated in
the Balbiani body (Figure 1D), suggesting it is rich in b sheet
structures, which is a hallmark of amyloids.
Xvelo Is Highly Concentrated in Balbiani Bodies
To analyze the composition of Balbiani bodies, we used quantitative mass spectrometry (McAlister et al., 2014; Wuhr et al.,
2014). This revealed that the most enriched Balbiani body proteins that are not part of organelles are Velo1 (commonly known
as Xvelo) and fetal hemoglobin subunits, Hba1 and Hbg1 (Figures S1AS1C). We focused on Xvelo because it is homologous
to zebrafish Bucky ball, which plays a crucial role in Balbiani
body organization (Bontems et al., 2009). Computational analysis of Xvelo sequence predicted an intrinsically disordered protein with no known domains, apart from a C-terminal positively
charged region that could bind RNA.
Quantitative western blotting using a peptide antibody against
the C terminus of Xvelo (Figure S2A) provided an estimate of
Xvelo
Mitochondria
Overlay
50 m
2 m
20 m
0.25 s
30 min
60 min
Xvelo-GFP
Prebleach
100
Intensity (A.U.)
Xvelo - WT
80
60
40
20
0
0
20
40
Time (min)
60
A
NH2
1
WT
82
398
F4
4D
NQPRPYFYAQP...GNPDDPDDSVAL
7D
NQPRPDDDAQP..GNPDDPDDSVAL
Fragment 3
Fragment 4
Xvelo-woF1
GFP
Fragment 2
779 aa
COOH
598
F3
F2
Prion-like domain
82
21
58
NQPRPYFYAQP...GNPYFPYYSVAL
Fragment 1
150
F1
Xvelo-WT
F1-7D
Xvelo-4D
F1-4D
Xvelo-7D
F1-WT
F1-7D
F1-WT
F1-4D
50 m
GFP
Ccy/CBb
0.8
50 m
t = 0.1 s
t = 10 min
0.2
0
GFP
Xvelo-WT
Zoom
F1-4D
F1- 4D
50 m
F1-7D
F1-7D
Fluorescence Recovery (% of initial)
0.4
F1-WT
F1-WT
Prebleach
0.6
100
80
WT
F1 - 4D
F1 - 7D
60
40
20
0
0
(C) mRNAs encoding for wild-type and PLD mutants of fragment 1-GFP were microinjected into oocytes. Oocytes were incubated overnight and imaged.
(D) Ratio of GFP concentration in the oocyte cytoplasm (Ccy) to the Balbiani body (CBb) in oocytes injected with mRNAs encoding for indicated proteins. Relative
concentrations were calculated by using oocyte or the Balbiani body volume from z stacks and the fluorescent intensity of GFP. Mean values and SEs of 10
oocytes are plotted.
(E) Internal rearrangement of fluorescent wild-type or mutant F1-GFP particles after photobleaching over time.
(F) The fluorescent recovery of photobleached wild-type or mutant F1-GFP in Balbiani bodies in (E) and two other biological replicates for each are shown by
quantification of fluorescence in bleached region over time normalized by an unbleached neighboring region.
(G) mRNAs encoding for full-length Xvelo-mCherry wild-type and GFP-tagged fragments (F1-WT-GFP, F1-4D-GFP, and F1-7D-GFP) were in vitro synthesized.
F1-WT-GFP or mutants were mixed with equal amounts of full-length Xvelo-mCherry mRNA and microinjected into the oocytes. After overnight incubation, the
oocytes were imaged by scanning confocal microscopy.
See also Figure S3.
Xvelo-WT
0 hour
1 hour
2 hours
3 hours
24 hours
98
62
49
38
28
Xvelo-7D-GFP
C
F1-WT-GFP
kDa
198
Xvelo-4D-GFP
Xvelo-WT-GFP
Xvelo-7D
Xvelo-4D
F1-WT
40 m
150
100
Xvelo-WT
F1-WT
Xvelo-4D
Xvelo-7D
50
0
0
24
Time (h)
Control
1 % SDS
0.1h
12h
24h
1000
Xvelo-WT
900
ThT (A.F.U)
800
40 m
700
600
500
400
300
F1 - WT
200
ThT
Blank
Mot3
Xvelo-7D
Xvelo-4D
F1- WT
Xvelo
Overlay
E
EB1
Xvelo-7D
Xvelo-4D
F1 - WT
Xvelo-WT
Xvelo-WT
100
-OC
mCherry
500
400
300
200
100
0
20 m
Ponceau
200
400
300
200
100
0
600
Oocyte st I, boiled
Oocyte stage I
Xvelo-WT
100nm
Xvelo-4D
400
SDS - PAGE
SDD - AGE
Egg extract
200
Distance (m)
Oocyte st I, boiled
600
ThT
10 m
400
500
-Xvelo
Ponceau
(E) Stage I oocytes were injected with mRNA coding for Xvelo-mCherry and incubated overnight. The oocytes were incubated in 10 mM ThT, washed twice, and
imaged by confocal microscopy. Bottom: zoomed in images. Line scans showing the co-localization of Xvelo-mCherry and ThT stain from five Balbiani bodies
were plotted. Each color represents the line scan of a different Balbiani body. We speculate that the outer rim Xvelo-mCherry signal belongs to the newly
translated Xvelo-mCherry protein that has just started to form a new, immature matrix and does not yet stain with ThT.
(F) SDD-AGE detects SDS-resistant Xvelo aggregates in vivo. Equal amounts of cytoplasmic extracts of stage I oocytes and mature eggs were loaded onto SDSPAGE. A five times more amount of egg extracts was loaded for SDD-AGE gels to make Xvelo concentrations comparable between the oocyte and egg extract
lanes. Xvelo was detected by an anti-Xvelo antibody.
See also Figure S5.
hnRNPA1
Tia1
Dazap1
FUS
FUS-156E
DIC
GFP
CPEB3
50 m
GFP
GFP
Xvelo-mCherry
Merge
Xvelo-mCherry
FUS(PLD)Xvelo
FUS
Buc(PLD)Xvelo
Buc
FUS/Xvelo
Buc /Xvelo
Buc
Prebleach
t = 0.1 s
t = 15min
D
Fluorescence Recovery (% of initial)
50 m
100
Buc
Buc(PLD)Xvelo
FUS(PLD)Xvelo
Xvelo - WT
80
60
40
20
0
0
200
400
600
Time (s)
800
1000
To further investigate the specificity of the Xvelo PLD for targeting to the Balbiani body, we swapped the PLD of Xvelo with
the PLD of FUS, an unrelated prion-like RNA binding protein,
and with the PLD of Bucky ball. The resulting chimeric proteins
were named FUS(PLD)Xvelo and Buc(PLD)Xvelo, respectively
(Figure S6C). Buc(PLD)Xvelo localized to the Balbiani body,
with a FRAP time in between Xvelo-WT and Bucky ball-WT (Figures 6C and 6D). FUS(PLD)Xvelo localized to the cytoplasm and
weakly to the Balbiani body (Figure 6B). The Balbiani body-localized protein recovered quickly after photobleaching, with a halflife of 1 min, much faster than that observed for Buc(PLD)Xvelo
(2 hr) (Figures 6C and 6D). Taken together, these data provide
strong evidence that the prion-like domains of functional homologs Xvelo and Bucky ball have unique features that target and
form a stable matrix in the Balbiani body.
We next examined the specificity of PLD interactions in vitro
with an aggregation assay that compared the assembly properties of Xvelo and FUS. We repeated previous reports showing
that FUS-WT forms liquid droplets in vitro, whereas the aggregation prone mutant FUS-156E forms aggregates (Patel et al.,
2015). We mixed Xvelo-RFP with either FUS-GFP or FUS156E-GFP in vitro in a high-arginine, high-salt buffer and then
diluted out the arginine and salt to initiate aggregation. XveloRFP networks and FUS-WT-GFP droplets or FUS-156E-GFP aggregates formed in the vicinity of each other but clearly were
separate and did not interact with one another (Figure S6D).
Thus, we conclude that PLDs do not aggregate with one another
randomly, even when they are in close proximity at high
concentration.
Xvelo Binds RNA and Clusters Mitochondria
A key biological function of Xvelo during Balbiani body formation
is likely to be the binding and concentration of organelles and
RNA through its amyloid-like self-assembly. We looked for direct
evidence that Xvelo can form a network that is sufficient to bind
and concentrate mitochondria and RNA. To test whether XveloGFP networks can bind RNA, we used a non-specific control
mRNA coding for mCherry and an RNA that is enriched in Balbiani bodies, the Xenopus homolog of nanos, xcat-2 (Zhou and
King, 1996). Both of the RNAs bound to the networks (only
mCherry-RNA results are shown). In contrast, the F1 fragment
that lacks the C-terminal motif did not bind to either of the
RNAs. Thus, Xvelo networks can sequester RNA in a manner
that requires a putative RNA-binding domain at the C terminus
of Xvelo (Figure 7A), but apparently without any strong RNA
sequence preference in vitro.
Germ cells receive a pool of organelles from cyst cells to form
Balbiani bodies and become oocytes in mice (Lei and Spradling,
2016). In early-stage oocyte development, these organelles are
(B) mRNAs encoding for Xvelo-mCherry and GFP-tagged Bucky ball (Buc), FUS, and the PLD-swap versions of Xvelo, in which the PLD of Xvelo was replaced
either by the PLD of Bucky ball (BucPLDXvelo) or the PLD of FUS (FUSPLDXvelo), were injected into the oocytes at equal concentrations and imaged after
overnight incubation.
(C) Internal rearrangement of fluorescent Bucky ball-GFP (Buc), and PLD-swap versions of Xvelo, Buc(PLD)Xvelo-GFP and FUS(PLD)Xvelo-GFP, after photobleaching over time.
(D) The fluorescent recovery of photobleached constructs in Balbiani bodies in (C), as well as Xvelo-WT and two other biological replicates for each, is shown by
quantification of fluorescence in a bleached region over time normalized by an unbleached neighboring region.
See also Figure S6.
RNA (546-14-UTP)
GFP
Mitochondria
Zoom/Merge
Control
Xvelo-WT
GFP
FUS-WT
F1- WT
10 m
FUS-156E
20 m
Mitochondria
Merge
GFP
Mitochondria
Merge
+ 2M KCl
Xvelo-WT
Egg Extract
Xvelo-WT
Xvelo-4D
GFP
20 m
5 m
Xvelo-WT + 2M KCl
Xvelo-WT
500
400
300
500
GFP
400
300
GFP
200
200
0
500
1000
1500
2000
500
1000
1500
2000
400
300
200
Mitochondria
1500
Distance (m)
2000
1000
1500
2000
500
Mitochondria
400
300
200
100
100
1000
500
Distance (m)
500
500
500
300
Distance (m)
Distance (m)
GFP
400
200
0
500
Xvelo-4D
Mitochondria
400
300
200
100
500
1000
1500
Distance (m)
2000
500
1000
1500
2000
Distance (m)
is organized by a functional amyloid into a dense matrix that sequesters mitochondria and other organelles.
The Balbiani body changes its properties during development:
it is a stable structure in the early oocyte, and it either disappears
in mammals (Hertig and Adams, 1967; Pepling et al., 2007) or
dissociates into small dispersed isles, called germ plasm, in
the mature oocytes of germ-plasm-containing species. This suggests that its formation and dispersal are regulated. We did not
detect any SDS-resistant Xvelo aggregates in egg extracts (Figure 5F), suggesting Xvelo does not form amyloid-like structures
in the egg. How this transformation occurs remains unclear. Our
preliminary data suggest that Xvelo is extensively phosphorylated in the egg, and most of these sites are not phosphorylated
in the oocyte. Regulation by phosphorylation could be a mechanism determining the dispersal of the Balbiani body, perhaps by
kinases or phosphatases that are activated at fertilization. Understanding the regulation of the physical state of Xvelo as the
oocyte matures will be important to elucidate the fate of the organelles residing in the Balbiani body.
Balbiani bodies are found in most vertebrates; mouse Balbiani
bodies were identified only recently, but Balbiani bodies in humans were observed decades ago, although they are almost untouched in the literature (Hertig and Adams, 1967; Pepling et al.,
2007). Could proteins similar to Xvelo be required for the formation of Balbiani-like structures in other organisms? In zebrafish,
bucky ball was identified in a maternal effect mutant screen as
the only gene that is essential for Balbiani body formation (Dosch
et al., 2004). Although the sequence similarity between Xvelo and
Bucky ball proteins are poor (Data S1B), these proteins have long
patches of intrinsically disordered regions, and score positive in
prion detection programs (Data S1C). Oskar is a key protein
required to organize Drosophila pole plasm (Ephrussi et al.,
1991), but no homologs of Oskar have been identified in vertebrates. We found that Oskar also is a disordered protein with a
predicted PLD (Data S1D). This suggests that amyloid-like selfassembly of a disordered protein could be an evolutionary
conserved mechanism for Balbiani body formation. Strikingly,
a recent paper has also linked the formation of large amyloidlike aggregates to gametogenesis in yeast, suggesting amyloid-like mechanisms may be involved in germline specification
across kingdoms (Berchowitz et al., 2015).
Despite the low sequence conservation of Xvelo and Bucky
ball (Data S1B), key residues in their PLDs are conserved, suggesting that these residues are structurally important and underlie the observed specificity of assembly. Xvelo does not interact
with other proteins with PLDs, such as FUS, upon self-assembly
in vitro. This is in contrast to the promiscuous behavior of many
disease-causing amyloids, which often show cross-seeding interactions and promote mutual nucleation events. We attribute
this to the fact that Xvelo self-assembly does not involve an intermediate liquid-like state. We speculate that fast assembly into an
inert amyloid-like structure prevents aberrant interactions with
other prion-like proteins, thus reducing the likelihood of a disease condition. We propose that rapid assembly kinetics and
high specificity are important driving forces underlying the evolution of functional amyloids.
What are the potential advantages of using an amyloid-like
mechanism to form the Balbiani body? One could imagine packing away germline components in amyloid-like structures is protective. The tightly packed structures could prevent other proteins from diffusing into them, such as regulators, thus keeping
the organelles in a dormant state. It could act to slow down the
diffusion of small toxic molecules generated by mitochondria,
which could be damaging. It also provides a novel way to organize the cytoplasm, forming a rigid, giant body, in which the organelles are clustered together into one place and kept there for
many years. Future studies are likely to provide mechanistic
insight into the central question of how the germline of an organism provides young cytoplasm with its complement of organelles
in every generation while the somatic cells age and die.
EXPERIMENTAL PROCEDURES
Detailed methods are available in Supplemental Experimental Procedures.
Oocyte Handling and Injections
All experiments using Xenopus and zebrafish were done with approval of the
Harvard Medical School (HMS) animal care review board. Ovaries were surgically removed from adult female Xenopus laevis frogs and treated with 2 mg/ml
collagenase 1A (Sigma) in 13 MMR by gentle rocking, until most of the oocytes
were clearly dissociated. Oocytes were later injected with mRNAs encoding
for indicated proteins by using a FemtoJet express microinjector (Eppendorf).
Xvelo Protein Purification from Insect Cells
Recombinant versions of MBP-Xvelo-GFP, -RFP, and no-fluorescent tag (for
negative-stain electron microscopy studies) were expressed in Sf9 insect cells
using the baculovirus expression system. Insect cells were harvested in lysis
buffer (50 mM HEPES [pH 7.6], 100 mM KCl, and 1 M arginine). The MBP
(maltose binding protein) tag was captured using dextrin Sepharose resin
and cleaved off using HRV 3C protease (MPI-CBG, in-house) by incubation
overnight on ice.
Microscopy
Differential interference contrast (DIC) and phase contrast microscopy for microinjections, perfusion chambers, and Balbiani body isolations were performed using a standard wide-field epifluorescence Nikon inverted microscope equipped with a Hamamatsu Orca CCD camera and 43, 103, and
203 dry objectives. Live confocal microscopy with oocytes was performed using Nikon A1R Laser Scanning confocal equipped with 103 dry and 403 water-immersion objectives. Spinning disc confocal images were taken with a Nikon Ti inverted microscope with Yokagawa CSU-X1 spinning disk confocal
with Spectral Applied Research Aurora Borealis modification, equipped with
203 dry, 403, and 603 oil-immersion objectives.
Semi-denaturing Detergent-Agarose Gel Electrophoresis
Stage I oocyte and egg extracts were prepared according to Field et al. (2014)
with intact actin. SDD-AGE was adapted from Alberti et al. (2009). The protein
concentrations of the lysates were adjusted and protein samples were mixed
with 43 sample buffer (80 mM Tris, 40 mM acetic acid, 2 mM EDTA, 20% [v/v]
(C) Line scans of Xvelo-WT-GFP and Xvelo-4D-GFP and mitochondria in Xenopus egg extracts. Five images were stitched together to have an area spanning
larger than 2 mm2. Each color represents a different field.
(D) Recombinant FUS-WT-GFP or the aggregation prone mutant, FUS-G156E-GFP, was added to Xenopus egg extracts with intact actin. Arrows point to the
exclusion zones of mitochondria in the presence of FUS structures.
See also Figure S7.
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at 90 V, followed by wet transfer to nitrocellulose membranes (Amersham Biosciences). Xvelo protein was detected by an anti-Xvelo antibody.
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Article
Authors
Salman F. Banani, Allyson M. Rice,
William B. Peeples, Yuan Lin,
Saumya Jain, Roy Parker,
Michael K. Rosen
Correspondence
michael.rosen@utsouthwestern.edu
In Brief
What are the general principles that
define the composition of phaseseparated cellular bodies?
Highlights
d
Article
Compositional Control
of Phase-Separated Cellular Bodies
Salman F. Banani,1 Allyson M. Rice,1 William B. Peeples,1 Yuan Lin,1 Saumya Jain,2 Roy Parker,2 and Michael K. Rosen1,*
1Department
of Biophysics and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA
of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA
*Correspondence: michael.rosen@utsouthwestern.edu
http://dx.doi.org/10.1016/j.cell.2016.06.010
2Department
SUMMARY
RNA binding proteins that are not important for P body assembly (Buchan and Parker, 2009). Within cellular bodies, clients
diffuse much more rapidly than scaffolds (Dundr et al., 2004;
Weidtkamp-Peters et al., 2008), suggesting that client-scaffold
interactions are more transient than the interactions among
scaffolds.
Compositional regulation is a general property of many cellular
bodies and may be crucial to their function. Cellular body compositions change during the phases of the cell cycle or in
response to stresses (Dellaire et al., 2006; Grousl et al., 2009).
Despite their importance, the fundamental principles governing
cellular body composition have been experimentally difficult to
elucidate, owing to the complex nature of both scaffolds and
clients and the diversity of species that reside within bodies.
However, simplified model systems composed of few types
of molecules, each with well-defined interaction elements, can
help isolate key molecular parameters and thus have the potential to reveal generalizable concepts.
Here, we describe the biochemical and cellular behavior of
three different sets of engineered molecules as simplified but
representative multivalent scaffolds and low valency clients,
which form model cellular bodies. Clients were differentially recruited into the bodies based on the relative stoichiometries of
the scaffolds. Changes in client recruitment occurred sharply
and on cellular timescales as the scaffold stoichiometries or valencies changed. Client partitioning also depended on client
valency. These findings lead to a simple mass action model
that predicts many features of the observed client partitioning
behavior and suggests how cellular body compositions could
be regulated in cells. Behaviors analogous to those of the model
systems were observed in PML NBs in mammalian nuclei and P
bodies in yeast cytoplasm. Thus, although natural cellular bodies
are complex, their compositions may be governed by simple underlying rules and could be altered based on parameters that are
easily tunable through cellular and evolutionary mechanisms.
RESULTS
Scaffold Stoichiometries Dictate Client Recruitment
We began by studying three independent pairs of interacting
multivalent scaffolds in vitro. These systems consisted of (1) a
protein with ten repeats of human SUMO3 (polySUMO) and a
protein with ten repeats of the SUMO Interaction Motif (SIM)
from PIASx (polySIM); (2) a protein with four repeats of the second SH3 domain from Nck (polySH3) and a protein containing
four repeats of a Proline-Rich Motif (PRM) from Abl1 (polyPRM)
(Li et al., 2012); and (3) the PTB protein (contains four RNA recognition motifs [RRMs]) and an RNA with five repeats of the RRM
recognition element UCUCU (polyUCUCU) (Li et al., 2012).
Each of these pairs phase separated when mixed together, but
not when individual components were alone in solution (Li
et al., 2012; Figure S1A; and data not shown).
To model client recruitment into the bodies, we engineered a
series of fluorescently labeled, monovalent clients (containing
a single element that binds the scaffold) and characterized their
partitioning into droplets generated by their cognate scaffolds.
We mixed (1) GFP-SUMO and RFP-SIM (or GFP-SIM) with
polySUMO/polySIM (Figure 1A); (2) GFP-PRM and RFP-SH3
652 Cell 166, 651663, July 28, 2016
A
GFP-SUMO
SIM Module
Concentration (M)
polySUMO + polySIM
RFP-SIM
Merge
90
80
70
60
50
50
60
70
80
90
50
60
70
80
90
50
60
70
80
90
Scale: 100 m
Solutions of multivalent scaffolds plus the indicated clients were imaged for client fluorescence.
AF, Alexa fluorophore.
(A) GFP-SUMO (green) and RFP-SIM (magenta)
(100 nM each) were mixed with the indicated
module concentrations of polySUMO and polySIM.
(B) GFP-PRM (green) and RFP-SH3 (magenta)
(200 nM each) were mixed with the indicated
module concentrations of polyPRM and polySH3.
(C) UCUCU-AF647 (green) and RFP-RRM
(magenta) (200 nM each) were mixed with the
indicated module concentrations of polyUCUCU
and PTB.
See also Figure S1.
polyPRM + polySH3
GFP-PRM
RFP-SH3
Merge
RRM Module
Concentration (M)
SH3 Module
Concentration (M)
polySUMO + polySIM
GFP-(SUMO)2
75
60
45
30
15
0
90
75
60
45
30
15
0
90
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
70
90
le
odu M)
50
OM
SUM tration (
cen
Con
60
75
60
45
30
15
0
90
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
70
80
90
Co
le
odu M)
50
OM
SUM tration (
cen
Con
60
70
80
90
75
60
45
30
15
0
90
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
le
odu M)
OM
SUM tration (
n
once
60
70
80
90
le
odu M)
50
OM
SUM tration (
cen
Con
60
GFP-(SIM)3
75
60
45
30
15
0
90
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
Co
Co
50
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
GFP-(SIM)2
Partition Coefficient
Partition Coefficient
GFP-(SIM)1
75
60
45
30
15
0
90
Co
Co
80
Partition Coefficient
S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)
Co
GFP-(SUMO)3
Partition Coefficient
Partition Coefficient
Partition Coefficient
GFP-(SUMO)1
50
70
80
90
le
odu M)
OM
SUM tration (
n
once
60
50
70
80
90
le
odu M)
OM
SUM tration (
n
once
60
polySIM
Merge
90
80
70
60
50
50
60
70
80
90
50
60
70
80
90
50
60
70
polySIM
150
120
90
60
30
0
90
90
50
80
70
le
60
odu M)
OM
SUM tration (
n
ce
Con
90
80
70
le
60
odu M)
M
O
SUM tration (
n
ce
Con
50
Droplet
Free Sites
100
Clients
100
60
80
70
70
80
60
90 SUMO
50 SIM
Bulk
polySUMO
polySIM
60
80
70
60
80
50
90 SUMO
50 SIM
Partition Coefficient
102
polySUMO
polySIM
Droplet/Bulk Ratio
100
80
60
40
20
0
50
90
S 80
nc IM 70
en M
tra od 60
tio ule 50
n
(
M
)
Droplet
80
60
40
20
polySUMO
polySIM
0
50
90
60
80
70
70
80
60
80
60
100
40
20
102
90 SUMO
50 SIM
0
50
90
60
80
70
70
80
60
3000
2500
2000
1500
1000
500
0
50
90
Bulk
Co
Co
Partition Coefficient
Partition Coefficient
150
120
90
60
30
0
90
S 80
nc IM 70
en M
tra od 60
tio ule 50
n
(
M
)
90
SUMO Client
80
Scale: 100 m
SIM Client
SIM Module
Concentration (M)
polySUMO
90 SUMO
50 SIM
Figure 4. Droplets Interchange Composition on Cellular Timescales without Compromising Structural Integrity
(A) Schematic of experiment. After equilibration of 100 nM GFP-SUMO and 100 nM RFP-SIM with polySUMO and polySIM at module concentrations of 60 mM and
80 mM, respectively, concentrations of the polySUMO and polySIM were abruptly shifted to 80 mM and 60 mM, respectively, for trajectory 1 and vice versa for
trajectory 2.
(B) Time lapse imaging of droplets starting immediately after the abrupt change in concentrations of polySUMO and polySIM, showing merged, pseudocolored
fluorescence signals from GFP-SUMO (green) and RFP-SIM (magenta). Note that small droplets (white arrows, top) interconvert more quickly than larger droplets
(bottom).
(C) 6 mM of a (SUMO)9-(SIM)8 scaffold containing Ulp1 cleavage sites after only the two N-terminal SUMOs was equilibrated with 50 nM of GFP-(SIM)2 (green) and
RFP-(SUMO)2 (magenta). Time lapse imaging was started immediately after addition of 10 nM of Ulp1. Pseudocolored images showing merged fluorescent
signals from the two clients are shown.
See also Figure S5 and Table S3.
C
(SUMO)5-(SIM)10
Scaffold:
Scale:
RFP-(SUMO)6-(SIM)10
YFP-SIM
YFP-SUMO
YFP-SIM
Scale:
D
12
Scaffold
Client
10
8
6
4
2
0
GFP-SUMO
GFP-SIM
Scaffold
Client
10
8
6
4
2
0
GFP-SUMO
GFP-SIM
RFP-(SUMO)10-(SIM)6
RFP-(SUMO)6-(SIM)10
14
YFP-SUMO
YFP-SIM
12
10
8
6
4
2
0
10
12
14
12
(SUMO)5-(SIM)10
Client Partition Coefficient
(SUMO)10-(SIM)5
Client Partition Coefficient
RFP-(SUMO)10-(SIM)6
YFP-SUMO
Scaffold
Client:
GFP-SIM
Client
Scaffold
Client
GFP-SUMO
Scaffold
GFP-SIM
Scaffold
(SUMO)10-(SIM)5
GFP-SUMO
Client
Client:
Client
Scaffold:
14
YFP-SUMO
YFP-SIM
12
10
8
6
4
2
0
10
12
14
DNA damage repair, apoptosis, and anti-viral responses (Lallemand-Breitenbach and de The, 2010). The PML protein appears
to be the primary scaffold for these bodies (Ishov et al., 1999).
PML can self-assemble via elements within its Tripartite Motif
(TRIM) (Antolini et al., 2003; Huang et al., 2014) and also via binding of its conserved SIM element to SUMOs conjugated at up to
eight sites in the protein (Nisole et al., 2013; Shen et al., 2006).
Though not strictly required for body assembly (Brand et al.,
2010; Sahin et al., 2014), SUMO-SIM interactions likely
contribute substantially to body architecture, as deletion of the
SIM motif or perturbations to PML SUMOylation via mutagenesis, viral infection, or knockdown/overexpression of SUMO
ligases/proteases can cause changes in the size, number,
morphology, or dynamics of PML NBs (Best et al., 2002; Hattersley et al., 2011; He et al., 2015; Muller and Dejean, 1999; Shen
et al., 2006; Weidtkamp-Peters et al., 2008). SUMO-SIM interactions also appear to be critical for the recruitment of many PML
NB clients (e.g., Daxx and Sp100) (Lin et al., 2006; Van Damme
et al., 2010; Zhong et al., 2000).
We initially examined partitioning of GFP-tagged SUMO/SIM
clients into endogenous PML NBs in U2OS cells (Figure S7A).
Immunofluorescence imaging using an antibody against PML revealed numerous PML NBs in nearly all cell nuclei. For each
Scaffold:
RFP(SIM)3
GFP-PML(SUMO)
Client:
RFP(SUMO)3
Strain:
RFP(SIM)3
Xrn1-GFP
WT
lsm1
dcp2
Scaffold
Client:
GFP-PML
RFP(SUMO)3
Merge
Client
Intensity Ratio
Scale:
2 m
Scale:
10 m
10
8
6
4
2
0
1.88
3.71
4.08
Median
Client
Intensity Ratio
**
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
1.20
***
2.79
2.14
***
1.21
Median
***
***
***
Figure 6. Client Recruitment into Natural Cellular Bodies Is Affected by Scaffold Stoichiometries
(A) Images of RFP-SUMO or RFP-SIM (red) co-transfected with GFP-PML or GFP-PML(SUMO) (green) into PML/ MEFs (top); nuclear staining with Hoecsht
33342 (blue). Plots (bottom) show IRs from individual cells (black dots) and median values (red horizontal lines). Each symbol represents the average IR (see
Experimental Procedures) for all puncta in a given cell. 3244 cells were analyzed per sample, each on average containing 16 or 5 puncta per cell with GFP-PML or
GFP-PML(SUMO), respectively. Distributions were statistically compared using the Wilcoxon rank sum test followed by the Bonferonni correction for multiple
comparisons to determine significance. ***p < 0.001. Dotted line, IR = 1.
(B) Representative images of WT, lsm1D, or dcp2D yeast strains carrying Xrn1-GFP (green) in their genomes (top). Distributions of Xrn1-GFP IRs (bottom), where
each symbol represents IR corresponding to an individual P body. 13 P bodies per cell were analyzed from a set of 410 cells per sample. Analysis for
significance was performed as in (A). **p < 0.01.
See also Figure S7.
C
E
ure 7E). Moreover, molecules with otherwise appropriate physicochemical properties (e.g., complementary charge) may also
partition into droplets due to non-specific interactions (Li et al.,
2012). Clients containing multiple types of interaction elements,
some mirroring scaffold-scaffold interactions and others not,
could show complex behaviors that are essentially superpositions of these individual effects. This reasoning may explain
the recruitment of Xrn1-GFP into P bodies without perturbation
of mRNA content (i.e., on what may be the non-cognate side
of the phase diagram diagonal), as well as the enhanced
recruitment when cellular mRNA is increased, as observed in
Figure 6B.
Complexities of Natural Cellular Bodies
Although natural cellular bodies are appreciably more complicated than our engineered model systems, their compositions
may still be understood with simple extensions to the framework
we present here. First, cellular bodies may have multiple scaffolds held together by different types of multivalent interactions.
For example, RNA granules likely have multiple scaffolds with
contributions from both low-complexity sequence elements,
as well as RNA and RNA-binding domains. PML NBs likely
assemble by a combination of TRIM and SUMO-SIM interactions. Multiple types of scaffolds and scaffold interactions
may cooperate to synergistically promote polymerization and
phase separation, as suggested previously (Lin et al., 2015).
Moreover, clients may also possess multiple classes of low
valency elements that can each interact with scaffolds. Nevertheless, in the absence of cooperativity, one can think of the
different interaction motifs independently. For any given class,
the corresponding free sites in a scaffold will dictate partitioning
of clients that can bind to those sites. Indeed, perturbing one
type of interaction motif within PML NBs or P bodies had strong
effects on the recruitment of clients that bind to that motif
(Figure 6).
Cell 166, 651663, July 28, 2016 659
Conclusion
We demonstrate how cellular body assembly, when driven by
heterotypic polymerization and concomitant phase separation,
naturally leads to a simple and predictive model for compositional control of these structures. Our model suggests how
bodies could be switched sharply between distinct compositional (and thus functional) states on a range of biological timescales. Moreover, it suggests that superficially similar cellular
bodies composed of a given set of scaffolds may be markedly
different in their composition and function, depending on the
relative scaffold stoichiometries. Thus, a complete understanding of cellular bodies may require knowing relative scaffold
amounts in addition to scaffold identities.
Our studies thus provide a mechanistic framework for studying the biochemical and regulatory function of cellular bodies
owing to properties not attributable to any individual molecule
but rather to those intrinsic to the macroscopic structure itself.
EXPERIMENTAL PROCEDURES
SUPPLEMENTAL INFORMATION
AUTHOR CONTRIBUTIONS
Conceptualization, S.F.B. and M.K.R.; methodology, S.F.B. and M.K.R.; investigation, S.F.B., A.R., W.P., Y.L., S.J.; writing, S.F.B., A.R., R.P., M.K.R.; supervision, R.P. and M.K.R.
ACKNOWLEDGMENTS
We thank Louie Kerr, Kate Luby-Phelps, and Abhijit Bugde for assistance
with imaging; Chad Brautigam and Thomas Scheuermann for assistance
with ITC; Mark Kittisopikul for helpful discussions regarding image analysis,
numerical fitting, and statistical testing; Pier Paolo Scaglioni for providing
PML/ cells; Rama Ranganathan for critical reading of the manuscript;
and members of the Rosen lab for helpful discussions. This work was
supported by the Howard Hughes Medical Institute, the HCIA program
of HHMI, grants from the NIH (R01-GM56322) and Welch Foundation
(I1544), a Sara and Frank McKnight Graduate Fellowship (to S.F.B.), and
the NSF (1000196079; to A.M.R.).
Received: September 8, 2015
Revised: March 13, 2016
Accepted: June 1, 2016
Published: June 30, 2016
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Article
Authors
Bernhard Hampoelz,
Marie-Therese Mackmull,
Pedro Machado, ..., Thomas Lecuit,
Yannick Schwab, Martin Beck
Correspondence
martin.beck@embl.de
In Brief
Rapidly growing embryos meet the
challenge of stocking the nuclear
envelope with nuclear pore complexes by
keeping a ready store of pores in stacked
membranous rings that feed into the
envelope without puncturing it.
Highlights
d
Article
Pre-assembled Nuclear Pores Insert
into the Nuclear Envelope
during Early Development
Bernhard Hampoelz,1 Marie-Therese Mackmull,1 Pedro Machado,2 Paolo Ronchi,2 Khanh Huy Bui,1,5 Nicole Schieber,4
Rachel Santarella-Mellwig,2 Aleksandar Necakov,1 Amparo Andres-Pons,1 Jean Marc Philippe,3 Thomas Lecuit,3
Yannick Schwab,2,4 and Martin Beck1,4,*
1European
Molecular Biology Laboratory, Structural and Computational Biology Unit, 69117 Heidelberg, Germany
Molecular Biology Laboratory, Electron Microscopy Core Facility, 69117 Heidelberg, Germany
3Aix-Marseille Universite
, CNRS, IBDM UMR 7288, 13009 Marseille, France
4European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, 69117 Heidelberg, Germany
5Present address: Department of Anatomy and Cell Biology, Groupe de Recherche Axe
sur la Structure des Proteines (GRASP), McGill
University, Montreal, Quebec H3A 0C7, Canada
*Correspondence: martin.beck@embl.de
http://dx.doi.org/10.1016/j.cell.2016.06.015
2European
SUMMARY
Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos,
NPCs reside not only within the NE, but also at
some endoplasmic reticulum (ER) membrane sheets,
termed annulate lamellae (AL). Although a role for AL
as NPC storage pools has been discussed, it remains
controversial whether and how they contribute to the
NPC density at the NE. Here, we show that AL insert
into the NE as the ER feeds rapid nuclear expansion
in Drosophila blastoderm embryos. We demonstrate
that NPCs within AL resemble pore scaffolds that
mature only upon insertion into the NE. We delineate
a topological model in which NE openings are critical
for AL uptake that nevertheless occurs without
compromising the permeability barrier of the NE.
We finally show that this unanticipated mode of
pore insertion is developmentally regulated and operates prior to gastrulation.
INTRODUCTION
In eukaryotes, the double membranous nuclear envelope (NE)
encloses the nucleoplasm and separates it from the cytoplasm.
The inner nuclear membrane (INM) provides contact with chromatin and the outer nuclear membrane (ONM) is continuous
with the endoplasmic reticulum (ER). The two bilayers are fused
at nuclear pore complexes (NPCs) that form aqueous channels
through which regulated transport of macromolecules occurs.
NPCs consist of multiple copies of 30 different nucleoporins
(Nups) that are organized into biochemically distinct sub-complexes (Figures S1A, S1A0 , and S1B). Two such modules, the inner ring complex (also called Nup93 complex) and the Y-complex (also called Nup107 complex) constitute the NPC scaffold
that is symmetric across the NE plane. FG-Nups (containing
phenylalanine-glycine rich intrinsically disordered protein domains) dock onto the scaffold. They constitute the permeability
barrier and interact with translocating cargo complexes. Some
of them (e.g., Nup214/88, Nup358 [RanBP2], and Nup153) introduce asymmetry by specifically binding to the cytoplasmic or nuclear face of the NPC, respectively (reviewed in Grossman et al.,
2012) (Figure S1B).
Obviously, the sheer size and compositional complexity of
NPCs renders its assembly and membrane insertion a very intricate task. Two distinct NPC assembly pathways that are temporally separated during the cell cycle have been described. First,
during interphase, NPCs are assembled de novo onto an enclosed NE (DAngelo et al., 2006). Interphase assembly occurs
ubiquitously throughout eukaryotes and strictly requires the
fusion of the INM and ONM by a mechanism that is only partially
understood (Doucet and Hetzer, 2010). Second, no membrane
fusion is required for NPC assembly at mitotic exit. This so-called
postmitotic assembly mode is restricted to eukaryotes that
disassemble their NPCs during mitosis into soluble sub-complexes after phosphorylation by mitotic kinases (Laurell et al.,
2011). In anaphase, de-phosphorylation of Nups is thought to
trigger the ordered re-assembly onto the separated chromatids
before or while membranes enclose daughter nuclei (Doucet
et al., 2010; Dultz and Ellenberg, 2010; Dultz et al., 2008). Both
insertion mechanisms rely on the stepwise recruitment of
pre-assembled sub-complexes. An insertion of pre-assembled
NPCs into the NE has (to the best of our knowledge) not yet
been described.
NPCs not only reside within the NE but are also found in
stacked cytoplasmic membranes termed annulate lamellae
(AL) that are a subdomain of the ER (Figure S1C) (Cordes
et al., 1996; Daigle et al., 2001). Based on two-dimensional
(2D) transmission electron micrographs these membrane stacks
have been perceived as parallel membrane sheets decorated
with NPCs (hereafter called AL-NPCs) that morphologically
appear similar to their counterparts on the nuclear envelope
(NE-NPCs) (Kessel, 1983). AL appear in some but not all transformed cell lines (Cordes et al., 1996; Daigle et al., 2001) and
are highly abundant in germ cells and early embryos throughout
664 Cell 166, 664678, July 28, 2016 2016 The Author(s). Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
(C) NPC density stays constant as nuclei grow. Quantification of normalized nuclear surface increase (blue curves) and the mean GFP::Nup107 fluorescence
intensity SD at the NE (red curves) during interphases. Values on both graphs are normalized to the earliest measured time point for each movie (n = 71 nuclei in
four embryos).
(DE) The Y-complex protein Nup107 and WGA-labeled transport Nups co-localize at the NE and at AL-NPCs that insert to the NE. Top view still (DD00 ) and
kymograph (E) from a time-lapse movie imaging a WGA-Alexa555 injected syncytial blastoderm embryo expressing GFP::Nup107 (see also Movie S2). Insertion is
captured in the kymograph (E) that spans the region of interest (ROI) boxed in (D).
(FG) Stills (FF00 ) and kymograph (G) of the blue shaded ROI from a time-lapse movie recording photo-converted EosFP::Seh-1 before (0 s, F) or after (6 s, F0 , and
145 s, F00 ) photo-conversion of AL-NPCs adjacent to the NE. NPC transfer from AL to the NE is documented by the lateral dispersion of the converted signal, which
starts after 100 s (G).
(H) AL-NPC number drops during early interphases. Quantification of the relative AL-NPC number inferred from GFP::Nup107 fluorescence for four embryos liveimaged over interphases. AL-NPCs were counted from 1 mm distant z sections spanning nuclear height in a constant field of view comprising 10 nuclei for each
embryo.
(I) GFP::Nup107 fluorescence intensity shifts from AL to the NE in the first 25% of interphases, when AL number drops the most (H). Fluorescence intensities were
integrated over consecutive confocal slices covering the entire nuclear height at the NE (NE-NPCs) and at AL (AL-NPCs). Cytoplasmic GFP::Nup107 was
determined from the mean fluorescence intensity. Analysis was done on two embryos (n = 13 and 11 nuclei, respectively). See Figure 1A for representative image;
error bars represent SD over multiple ROIs. All images in Figure 1 are acquired from embryos in cycles 1013 of the syncytial blastoderm stage.
See also Figure S1 and Movies S2 and S4.
(CD00 ) Top views onto a fixed Drosophila syncytial blastoderm embryo in interphase. The nuclear basket components Nup153 (C and C0 ) and Mtor (D and D0 ) are
absent from AL-NPCs (arrowheads in C00 , D00 ), which stain positive for mAb414 (C and C00 ) or WGA (D and D00 ), both labeling FG-Nups (including Nup358).
(E and E0 ) AL-NPCs (E) are pore scaffolds made of transmembrane Nups, the inner ring, and Y-complex nucleoporins, extended by Nup358, which might or might
not be attached symmetrically across ER membranes. NE-NPCs (E0 ) recruit soluble Nups to construct the mature pore that is asymmetric across the NE.
See also Figures S3 and S4.
DISCUSSION
Collectively, the following scenario emerges from our data. AL
are abundant in early Drosophila embryos and predominantly
contribute to maintain the constant NE-NPC density in the expanding NE during interphase. The abundance of AL at the
cortical nuclei layer thereby oscillates together with the progression of the consecutive interphases until the start of global transcription when AL disappear and the mode of NPC insertion
changes (Figures 7A and 7B). During each onset of early interphases, AL-NPCs are assembled similarly to NE-NPCs but since
the combined nuclear surface of the two daughter nuclei is
smaller as compared to the parental nucleus, they remain in the cytoplasm. As interphases progress, AL-NPCs feed into
the pool of NE-NPCs alongside ER membranes that augment NE surface during
rapid nuclear expansion (Figure 7C). AL
insertion is enabled by NE openings that
might either persist from previous mitosis
or form de novo by an unknown mechanism (Figure 7D). Upon AL insertion, the
NE permeability barrier remains unperturbed, likely because the NE openings
are entirely surrounded by the ER
network. The inserting NPCs comprise
pre-assembled NPC scaffolds that recruit
the full set of Nups only subsequent to
insertion and only then establish transport
competence.
Why do the expanding nuclei of the syncytial blastoderm maintain a constant
number of NPCs per surface area despite
their transcriptional inactivity? One might
surmise that this is due to mechanical
properties but also temporal constraints. The insertion of NPCs
might be crucial to enable the massive influx of material into
the nucleoplasm during nuclear expansion (volume increase).
Indeed, the strained configuration of nuclei is reflected by their
strong mechanical response (NE tumbling) upon disruption of
the NE and permeability barrier after laser puncture. Second,
the batch transfer of entire NPC scaffolds as inherent parts of
membrane sheets overcomes the described kinetic constrains
of interphase assembly in mammalian cells, that are not compatible with the short interphases in the Drosophila syncytium (Dultz
and Ellenberg, 2010). Given the abundance of AL-NPCs and the
reported high insertion rate of NPCs into the NE of Xenopus
Cell 166, 664678, July 28, 2016 675
microscopy (EMCF), and proteomic (PCF) core facilities and Petra Riedinger
for graphical design. We are grateful to Drs. P. Paul-Gilloteaux, X. Heiligenstein, and S. Mosalaganti for assistance and expertise in correlative light
and electron microscopy analysis. B.H. was supported by the Agence Nationale de la Recherche (ANR) (Programme Blanc) NeMo (to T.L) and the European Molecular Biology Organisation (EMBO). K.H.B. was supported by postdoctoral fellowships from the Swiss National Science Foundation (SNF),
EMBO, and Marie Curie Actions. M.B. acknowledges funding by EMBL and
the European Research Council (309271-NPCAtlas).
Received: October 25, 2015
Revised: April 15, 2016
Accepted: June 3, 2016
Published: July 7, 2016
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Article
Authors
Caitlin E. Gamble, Christina E. Brule,
Kimberly M. Dean, Stanley Fields,
Elizabeth J. Grayhack
Correspondence
fields@uw.edu (S.F.),
elizabeth_grayhack@urmc.rochester.edu
(E.J.G.)
In Brief
Rather than protein synthesis relying
solely on readout of individual codons,
pairs of codons dictate translational
efficiency, suggesting unexpected
coupling between tRNA binding sites
within the ribosome.
Highlights
d
Article
Adjacent Codons Act in Concert to
Modulate Translation Efficiency in Yeast
Caitlin E. Gamble,1,2,6 Christina E. Brule,3,4,6 Kimberly M. Dean,3,4,8 Stanley Fields,1,5,7,* and Elizabeth J. Grayhack3,4,7,*
1Departments
SUMMARY
reverse of each codon pair, and the two individual codons would
be represented many times in different contexts.
To detect differences in GFP expression, we used fluorescence-activated cell sorting (FACS) to separate yeast cells into
three fluorescence bins. For the (NNN)3 library, we made and
separately sorted two independent yeast libraries. We estimated
that the assay detected expression levels, relative to a no-insert
GFP reference, from 75%100% in bin 1, from 25%75% in
bin 2 (median 43% of bin 1 median), and from 2.5%25% in bin 3
(median 6% of bin 1 median) (Figure 1A); GFP variants with stop
codons migrated into the background bin. Following FACS, we
sequenced the three-codon insertions from cells in each bin,
carried out quality filtering, and determined the relative distribution of sequences. We estimated mean expression (GFPSEQ) for
each sequence based on the sequences distribution across
bins and applying the median fluorescence of a bin to all reads
counts in that bin. GFPSEQ scores correlated across the three libraries (r = 0.91 to 0.93) (Figure S1A) and with mean GFP expression of 76 individual constructs measured by flow cytometry
(GFPFLOW) (r = 0.81), although binning limited the resolution
(Figure S1B).
We considered that amino acid sequences encoded by the insertions could affect GFP stability or expression, although most
of these effects should be mitigated by using superfolder GFP,
which has robust fluorescence even when fused to several insoluble proteins (Pedelacq et al., 2006). Thus, for downstream analysis, we included only the 35,811 unique DNA sequences
specifying one of 5,148 tripeptides that had at least one synonymous sequence above the mean of all GFPSEQ scores (which
left out 4.1% of tripeptides and 6.2% of DNA variants). For
each DNA sequence, the highest scoring sequence encoding a
synonymous peptide served as its synonymous reference. We
scored expression due to codon usage (syn-GFPSEQ) as the
GFPSEQ ratio of a given sequence and its synonymous reference.
As expected, most synonymous variants had similar expression (Figure 1B; Table S1), with a mean syn-GFPSEQ of 0.954.
However, 1,119 DNA sequences (low variants) had syn-GFPSEQ
ranging from 0.059 through 0.647 (three SDs or more below the
mean). Intermediate variants comprised 5,127 sequences from
0.648 through 0.953; high variants comprised 24,417 non-reference (as well as 5,148 reference) sequences with syn-GFPSEQ
greater than 0.953.
There were no examples in which the use of an individual
codon consistently reduced expression to a degree detectable
in our assay. The median syn-GFPSEQ for each set of variants
containing one or more copies of a given codon ranged from
0.97 to 1.00, but the use of broad expression bins limited our
ability to detect differences in GFPFLOW values between 75%
and 100% of the reference GFP. A subset of codons occurred
frequently in low variants (Table S2), suggesting that combined
use of particular codons may dramatically reduce expression.
To identify inhibitory codon pair candidates, we looked for
combinations of adjacent codons enriched in the low-variant
category. We found 293 six-base sequences (non-gapped
6-mers) enriched in the low variants at one or more of the four
possible starting positions of the nine-base insertions (permutation p value % 0.001; Table S3). Most six-base sequences were
enriched at a single position, as might be expected if they form
C
1. Library of GFP variants
Library insertion
PGAL1,10
RFP
GFP
RFP
6-mer position
NNN-NNN-NNN
2
3
4
5
4
105
3
104
2
103
104
105
GFP
3. High throughput sequencing of bins
B
10000
High
Intermediate
Low
7500
500
5000
250
0
-log10(p-value)
DNA variants
750
2500
0.00
0
0.00
0.25
0.25
0.50
10
0.95
0.50
0.75
syn-GFPSEQ
Inhibitory
5
0.75
Arg-Pro
Optimal
1.00
CUU-CAG
CU U-CGG
GU A-CCG *
GU A-CGG
GUG-CGA *
GU A-CGA *
CG A-GCG *
CG A-CGG *
CG A-CGA *
CG A-CUG *
CG A-CCG *
CUG-GCG
CG A-AU A *
CUG-CCG *
CUG-CGA *
CU C-CCG *
CU U-CUG *
A U A-CGG *
A GG-CGA *
A U A-CGA *
CG A-CAU
A GG-CGG *
C U C-A U A *
CUG-CUG *
104
n=21
AGG-CGA* n=30
CUG-CUG
CUU-CUG
GUA-CCG*
GUA-CGA*
GUG-CGA*
0.00
0.4
0.2
0.0
D
Inhibitory pair
Single codon optimized
Insert
RFP PGAL GFP
0.25
0.50
0.75
Variant syn-GFP
1.0
0.8
0.6
0.4
0.2
0.0
1.00
SEQ
from 14%76% that of synonymous optimized variants (Figure 1D; Table S4).
Codon Pairs Mediate Frame-Dependent Inhibition in
Different Sequence Contexts
To assess the likelihood that inhibition is mediated by translation,
we examined the properties of the candidate pairs. If inhibition
were coupled to translation, then the enriched 6-base sequences would likely inhibit expression only when the two
codons were in-frame, but not when out-of-frame, where mechanisms decoupled from translation might explain enrichment.
For the 20 candidates, we compared the syn-GFPSEQ distribution of variants with these candidates at in-frame positions (the
six-base sequences starting at positions 1 and 4) (Figure 2A,
blue) to that of variants with the candidates at out-of-frame positions (the six-base sequences starting at positions 2 and 3)
682 Cell 166, 679690, July 28, 2016
Insert
RFP PGAL Rluc GFP
1.0
0.8
0.6
0.4
0.2
0.0
G
AAG CC
A- G
C CC
G
A- G
C
C
A
CUG-CGA*
0.6
CUG-CCG*
1.0
GFPFLOW ratio
(Inhibitory/Optimal)
CUG-AUA*
GFP
0.8
C
G
-C
C
U
C G
-C
C
A
CUC-CCG*
Rluc
CUC-AUA
1.00
Insert
-C
CGA-GCG*
0.75
RFP PGAL
CGA-CUG*
0.50
CGA-CGG*
0.25
Variant syn-GFPSEQ
AG
G
CG -CG
A A
CG -AU
A A
CG -CC
A G
CG -CG
A A
CG -CG
A G
CG -CU
A G
CU -GC
C G
CU - C C
G G
CU -CC
G G
GU -CG
A A
GU -CG
G- A
CG
A
CGA-CGA*
0.27
0.00
CGA-CCG*
0.65
0.49
n = 30
CGA-AUA*
CGA-GCG
Arg-Ala
1.0
0.8
0.6
0.4
0.2
0.0
AUA-CGG*
GFPFLOW ratio
(Inhibitory/Optimal)
AUA-CGA*
n=29
n=25
n=36
n=47
n=18
n=11
n=17
n=21
n=27
n=30
n=5
n=27
n=17
n=15
n=22
n=14
n=17
n=25
n=16
n=29
n=38
n=18
n=16
n=21
n=15
n=26
n=30
n=9
n=6
n=12
n=3
n=7
n=15
n=9
n=11
n=22
n=17
n=11
n=30
n=22
n=12
n=25
n=15
n=16
n=25
n=14
n=12
n=27
n=15
n=18
n=25
n=27
n=20
n=36
n=20
n=21
n=30
n=26
GFPFLOW ratio
(Inhibitory/Optimal)
AGG-CGG*
Out-of-frame
In-frame
Separated
Codon pair:
GFPFLOW ratio
(Inhibitory/Optimal)
or
IA wobble
UG wobble
Codon
Pair
R-R
R-R
I-R
I-R
R-I
R-P
R-R
R-R
R-L
R-A
L-P
L-I
L-P
L-R
V-P
V-R
V-R
AGG-CGA
AGG-CGG
AUA-CGA
AUA-CGG
CGA-AUA
CGA-CCG
CGA-CGA
CGA-CGG
CGA-CUG
CGA-GCG
CUC-CCG
CUG-AUA
CUG-CCG
CUG-CGA
GUA-CCG
GUA-CGA
GUG-CGA
CGA-CCG
105
Arg-Pro
Optimal
Pair
Vector
13.8 0.5
103
105 AGA-CCA
Arg-Pro
tP(CGG)*
87.6 2.4
Vector
33.1 1.6
AGA-GCU
Arg-Ala
tP(CGG)*
63.4 4.0
104
105
Vector
119.4 4.6
103
GFP
Vector
1.6
3 native tRNA
0.4
ND
AG
GCG
A
CG
AAU
A
CG
ACC
G
CG
ACG
A
CG
ACG
G
CG
ACU
G
CG
AGC
G
CU
CCC
G
CU
GCC
G
CU
GCG
A
GU
ACG
A
GU
GCG
A
NA
3 non-native tRNA
0.8
NA
tA(UGC)
118.9 5.3
104
105
GFP
1.2
0.0
tA(UGC)
67.3 8.6
104
Vector
103 76.5 0.5
103
C
GFPFLOW ratio
(Inhibitory/Optimal)
CGA-GCG
Arg-Ala
Inhibitory
104
Pair
RFP
Amino
Acid
Vector
3 non-native tRNA (decodes Pro CCG)
3 native tRNA (decodes Ala GCG)
CGA-GCG (Figure 2B) and three containing CGA-CGG (Figure S2A), the ratio of inhibitory to optimal GFPFLOW scores was
always less than 0.66. Thus, the codon pair was inhibitory in
different contexts, although the magnitude of inhibition varied.
This variation could reflect effects from RNA structure or additional sequence context (nucleotide, codon, or amino acid).
Additionally, each three-codon insert introduces four codon
pairs (including the invariant codons at positions 5 and 9), all of
which could affect translation.
If inhibition by codon pairs is a general function of their translation by the ribosome, then the codon pairs should reduce
expression when positioned at diverse locations within the coding sequence. However, the magnitude by which codons affect
expression can depend on their location relative to the start of
translation; for example, CGA codon repeats are more inhibitory
near the start of the coding sequence (Letzring et al., 2010; Wolf
and Grayhack, 2015). Therefore, we tested whether inhibition occurs at internal locations by inserting three copies of an inhibitory
codon pair at amino acid 100 (between an N-terminal GLN4(1-99)
domain and GFP) and at amino acid 318 (between Renilla luciferase and GFP) (Letzring et al., 2010; Wolf and Grayhack,
2015); we carried out this test for the 12 pairs with the lowest
syn-GFPSEQ medians. In each case, GFPFLOW with the inhibitory
pairs was lower than with optimized pairs (from 20%67%; Figure 2C). We also showed that increasing the copy number of the
codon pairs results in greater inhibition (three pairs tested at
amino acid 6 and two pairs tested at amino acid 100) (Fig-
5 native tRNA
0.8
0.6
0.4
0.2
NA
0.0
NA
NA
0.4
0.2
Lane 1 2 3 4 5
Inhibitory pair
Reverse order pair
1.2
n=34
AGG-CGA* n=30
AUA-CGG* n=22
n=27
CGA-CCG* n=16
n=22
CGA-CGG* n=22
n=38
0.8
CGA-GCG* n=18
n=30
0.4
CUC-CCG* n=16
n=15
0.0
CUG-AUA* n=22
CU
C
CC -C
G- CG
C U -A
C - CU
AC
AC
U
UAC C
U- UC
CC -C
G- CG
CU
C
A
G
-C
UG
G
-C
UA
A
-C
G
CU
CG
CG
A-
CG
G
A-
CG
Inhibitory pair
AGG-CGG* n=40
n=36
CC
A-
CG
CG
A-
AU
0.0
CG
1/5x
0.6
A-
1x
Phe(GAA) Arg(ICG)
1x 1x 1/5x 1x
tR(ICG) 2 leu2-d
Vector
0.8
CG
2 leu2-d
vector
tR(ICG)
tR(ICG)
Deacyl.
vector
tR(ICG)
tR(ICG)
2 LEU2
GFPFLOW ratio
(Inhibitory/Optimal)
5 non-native tRNA
1.0
GFPFLOW ratio
(Inhibitory/Optimal)
Vector
AG
GCG
A
CG
AAU
A
CG
ACC
G
CG
ACG
A
CG
ACG
G
CG
ACU
G
CG
AGC
G
CU
CCC
G
CU
GCC
G
CU
GCG
A
GU
ACG
A
GU
GCG
A
GFPFLOW ratio
(Inhibitory/Optimal)
n=22
CUG-CCG* n=28
n=30
N.D.
n = 38
Yeast ORFs
Q4
n = 98
Inhibitory pairs
n = 1,868
Q3
n = 398
Q1
n = 621
68%
32%
Q2
n = 713
No inhibitory pairs
n = 4,049
C
Inhibitory pairs
Reverse order pairs
Inhibitory pairs
No inhibitory pairs
10
**
*
**
*
**
*
log2(protein/mRNA)
log2(protein/mRNA)
10
n = 708
n = 118
n = 92
n = 335
n = 304
n = 477
n = 517
n = 308
n = 570
n = 111
n = 390
n = 39
n = 279
n = 678
**
*
0.05
CGA-GCG
Arg-Ala
n=35
CG
0.10
Ribosome occupancy
0.00
0.10
0.05
CUC-CCG
Leu-Pro
n=60
0.00
0.10
0.05
CGA-CUG
Arg-Leu
n=91
0.00
0.10
0.05
CUG-AUA
Leu-Ile
n=559
A-
G
CC
Mean
CG
G
A- G
A
CG CC
AU
C
AU
G
C
C
GA
-C
2 sd
3 sd
Inhibitory pair
Other codon pair
0.10
CG
CG
GA
A A
C
CG G
G
A- C-C
G
CC
U
U
U
A
A-C G G C CG
U
A
C
G
GCGG-C A
G
GU
CU -C
G
GA
CU
G
A A-C
CG
GG
G
AAU
UA G-C
-C
U
A
G
A
G
AG
AG
CU
0.05
0.00
0.00
0 +25 +50
-25
codon distance
-50
0.4
0.6
0.8
syn-GFPSEQ median
1.0
C
Inhibitory pair
Synonymous pair
Mean
UA*
GA
CU
Leu-Arg
A
CG G
AUAACG
AU
3 sd
Leu-Pro
0.05
2 sd
G*
CC
CUC CCG*
CUG
Leu-Ile
0.10
Ile-Arg
CG
CUG
0.00
*
UG
AC
CG
CG
AC
Arg-Pro
0.05
UA*
AA
CG
Arg-Leu
AG
CG
Arg-Ile
CG
0.10
Arg-Ala
CG
0.00
0.05
GA*
AC G*
CG ACG
G
CG
CG GA
AGGGGC
A
10 20 30
CG
C
GUA
Val-Pro
0.10
Ribosome occupancy
0.00
0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00
10 20 30
Rank
Reverse order pair
10 20 30
Inhibitory pair
1, E site E, P site P, A site A site, +1
0.10
Arg-Arg
0.15
Val-Arg
ity control, we required each read to have accurately called six bases
(AACGCA) immediately downstream of our variable region and for each of
the nine variable base calls to have a score of Q30 or better. To compare
read counts across bins, we corrected for the number of cell sorting events
in a given bin. See additional filtering and scoring details in the Supplemental
Experimental Procedures.
Analysis of Ribosome Profiling Datasets
We analyzed a whole-cell ribosome profiling sample with no cycloheximide
treatment from Jan et al. (2014). A-site codon footprint tallies were provided
by Jeff Hussman as described in Hussmann et al. (2015). See additional details
in the Supplemental Experimental Procedures.
Acidic Northern Blot Analysis
Bulk RNA, prepared from 3 OD pellets, was resolved on 6.5% acrylamide
gels at pH 5 as described previously (Alexandrov et al., 2006).
Explanation of the Statistical Methods
To assess the significance of each 6-mer sequences enrichment in low GFP
variants, we tracked occurrences of the 6-mer in low variants across
100,000 permutations. Variants were assigned to one of ten pools based on
GC count, and we shuffled the expression categories within each pool. From
this analysis we derived p values for the frequency of each 6-mer in low variants, based on the probability of obtaining as many, or more, low-variant
counts by chance.
We also estimated the chance probabilities of footprint densities. For each
pair, we carried out 10,000 permutations, in which we shuffled the A-site
codon footprint counts within each ORF with the pair and recalculated footprint density at ribosomal site positions. To directly evaluate the significance
of differences between synonymous pairs, we performed one-sided Fishers
exact tests on 2 3 2 contingency tables with the footprint counts for each
pair at ribosome site positions and at the remainder of codon positions within
a 100-codon window. See additional details in the Supplemental Experimental
Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.05.070.
AUTHOR CONTRIBUTIONS
C.E.G., C.E.B, S.F., and E.J.G. wrote the manuscript. C.E.G. and C.E.B. acquired data and performed the computational and experimental analyses,
respectively; K.M.D. identified tRNA suppressible variants from a pilot screen;
and E.J.G. and S.F. supervised the work.
ACKNOWLEDGMENTS
We thank Eric Phizicky, Andrew Wolf, Scott Butler, Gloria Culver, Adam Geballe, David Mathews, David Morris, and Yi-Tao Yu for discussions and comments on the manuscript, Jeffrey Hussman for assistance with ribosome
profiling data, Josh Hatfield, Shannon Schmitt, Blake Bentley, and Erin Eidschun for assistance with experiments, XiaoJu Zhang and David Mathews
for initial help analyzing codon use in the yeast genome, the URMC Flow Cytometry Resource and NCCR (1S10RR029229901) for technical support. This
work was supported by an NSF grant (MCB-1329545) (to E.J.G.) and an NIH
grant (1P41 GM103533) (to S.F.). C.E.B. was also supported by an NIH T32
Training Grant (GM068411), and C.E.G. was supported by an NSF Fellowship.
S.F. is an investigator of the Howard Hughes Medical Institute.
Received: December 18, 2015
Revised: April 14, 2016
Accepted: May 19, 2016
Published: June 30, 2016
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Article
Authors
Estienne Carl Swart, Valentina Serra,
Giulio Petroni, Mariusz Nowacki
Correspondence
mariusz.nowacki@izb.unibe.ch
In Brief
In some ciliates, all three stop codons
can either terminate translation or code
for an amino acid. Ribosomes may
interpret this ambiguity using
downstream features in the transcript,
indicating that translational termination
can be context-dependent.
Highlights
d
Article
Genetic Codes with No Dedicated Stop Codon:
Context-Dependent Translation Termination
Estienne Carl Swart,1 Valentina Serra,2 Giulio Petroni,2 and Mariusz Nowacki1,*
1Institute
SUMMARY
Cell 166, 691702, July 28, 2016 2016 The Author(s). Published by Elsevier Inc. 691
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
lation termination, and the typical histone H4 C-terminus may occasionally be extended by one or more amino acids.
While readthrough is conventionally classified as translation of
stop codons by near-cognate tRNAs, in C. magnum, which has
stop cognate tRNAs (see next section), translation through
stop codons by near-cognate tRNAs is effectively indistinguishable from translation by cognate tRNAs in ribo-seq data. Therefore, for the sake of simplicity, in C. magnum, we classify
readthrough as translation through codons that typically trigger
translation termination (as for H4.1d). It should be noted that in
C. magnum, multiple translation termination opportunities often
exist before the ribosome translates into poly(A) tails (on average
approximately five codons intervene between the primary and
additional downstream non-primary stops). As a consequence,
if extensions result from readthrough they are typically expected
to be very short. Even though multiple possible stop codons
exist, examples of imprecise termination as in H4.1d are in the
minority: 90% of transcripts examined with >20 RPFs situated
at their stops show no readthrough. Thus, overall readthrough is
quite low, e.g., a mean of <1.8% and median of 0% (Figure S3K).
The small amount of readthrough that does occur is most
readily detected when the ribosome occupies downstream
stops (Figure 3E).
Multiple lines of evidence therefore demonstrate that stop
codons as a class in the C. magnum and Parduczia sp. genetic
codes are ambiguous, whereas their individual codons are typically recognized unambiguously as either sense or stops, solving
the translation termination paradox.
In Search of tRNAs that Enable Stop Codon
Translation
All model ciliates have suppressor tRNAs that are complementary to and permit translation of reassigned stop codons (Eisen
et al., 2006; Hanyu et al., 1986; Kuchino et al., 1985). Although
we found a comprehensive set of tRNAs in our C. magnum
genome assemblies, including glutamine tRNAs capable of
recognizing UAA and UAG codons (Figures 4A and 4B; Data
S1G), we were unable to detect tRNATrps with UCA anticodons.
Given the high sequence coverage of the C. magnum macronuclear genome, it is unlikely that we missed tRNATrp(UCA)s. Ciliates possess both a micronuclear and a macronuclear genome,
with the former predominantly unsequenced in our C. magnum
assembly due to its comparatively low ploidy. It is also unlikely
that tRNATrp(UCA)s have gone undetected because they are micronuclear genome-encoded: although these genomes are transcriptionally active during ciliate sexual development they are
generally inactive during vegetative growth (Chen et al., 2014;
Nowacki et al., 2009) when many transcripts with UGA tryptophan codons are expressed. To test if CCA / UCA anticodon
editing produces a UGA-cognate tRNATrp, we sequenced RTPCR products targeting nuclear genome-encoded tRNATrps
and examined tRNA reads from small RNA sequencing data,
but found no signs of significant anticodon editing (see Supplemental Experimental Procedures).
All sequenced ciliate mitochondrial genomes encode a UGAcognate tRNATrp(UCA) (Swart et al., 2013) and so does that
of C. magnum (Figure S4A). Experiments in cell-free lysates
show cytoplasmic ribosomes can use yeast mitochondrial
Cell 166, 691702, July 28, 2016 693
Figure 2. Stop Codons in C. magnum and Parduczia sp.: Either Sense or Stop Codons
(A) C. magnum protein kinase alignment region highlighting putative sense stop codons. Standard genetic code stop codons are shown with stars, with
larger stars for UGA. MMETSP0210 IDs: CAMNT_0008311047, CAMNT_0008316317, CAMNT_0008295895, CAMNT_0008281491, CAMNT_0008274923,
CAMNT_0008274561, CAMNT_0008271577, CAMNT_0008291651, CAMNT_0008280967, CAMNT_0008289329.
(B) Ribosome-protected fragments (RPFs) mapped to a C. magnum tryptophan-tRNA ligase transcript (Data S1AC and S1AD). RPF coverage is calculated from
all the bases of 2532 nt RPFs.
(C) Histone H4 C-termini and stop codons (gray arrow, coding sequence) from C. magnum, Parduczia sp., and Homo sapiens. Poly(A) tails are visible at
C. magnum and Parduczia sp. mRNA 30 termini. Histone H4.1a H4.1d: MMETSP0210 IDs: CAMNT_0008274265, CAMNT_0008297091, CAMNT_0008284521,
and CAMNT_0008296393; Parduczia sp. histone H4 is MMETSP137 CAMNT_0047598059. H. sapiens histone H4 is GenBank: M16707.1. Judging from pairedend read mapping, the 30 UTR of H4.1a is incorrectly fused to a downstream transcript.
(D) RPFs mapped to histone H4.1c (Data S1AE and S1AF).
See also Figure S2.
necessary to translate all the stop codons are exceedingly unlikely to arise.
Given the existence of transcripts without 30 UTRs, we deduce
these regions are not essential for translation termination, and
we propose that the close proximity of a poly(A) tail and
poly(A)-interacting proteins, in particular PABPs, alone may be
necessary to trigger termination. Three prior observations favor
this hypothesis: (1) PABP overexpression enhances translation
termination when it is weak, implying that PABPs may be
involved in translation termination (Cosson et al., 2002), (2) tethering of a PABP 3773 nt downstream of a premature stop codon
substantially decreases NMD and results in recruitment of the
translation termination factor eRF3, suggesting that PABP is
involved in discriminating stops from premature stops (Amrani
et al., 2004); and (3) PABPs bind to AU-rich RNA including 30
UTRs (Baejen et al., 2014; Kini et al., 2016; Sladic et al., 2004).
Reassigned stop codons in C. magnum and Parduczia sp.
differ from conventional readthrough stops in standard genetic
code organisms because they are efficiently translated and
distributed throughout coding sequences, whereas conventional
readthrough stops are the major termination signals whose
disregard gives rise to modest levels of short protein extensions
(Dunn et al., 2013; Jungreis et al., 2011). From their distribution
throughout coding sequences, it is evident that most reassigned
codons in ciliates arose from substitutions of codons that were
already normally translated, rather than from readthrough stop
codons. Upon acquisition of a stop cognate tRNA, a shift in balance from translation termination to readthrough at stop codons
is expected. Normally this acquisition would immediately be
deleterious, due to the creation of aberrant C-terminal peptide
signals or the triggering of non-stop mRNA decay (Frischmeyer
et al., 2002) upon translation into mRNA poly(A) tails. By enforcing proper translation termination close to transcript ends, ciliates with ambiguous genetic codes provide a way of getting
around these problems.
Given that we detected no new genetic codes in 265 diverse
non-ciliate eukaryotic species from MMETSP, the abundance
of alternative genetic codes within ciliates is all the more striking.
Two hypotheses for the origin of genetic codes in ciliates are that
they were enabled by codon capture or eRF1 mutations. Under
the codon capture hypothesis (Osawa and Jukes, 1989)
when a codon disappears in a genome due to strong mutational
biases it may then be reassigned when a suitable cognate tRNA
arises (via tRNA duplication and anticodon mutation) and the
codon subsequently reappears. To date, all sequenced ciliate
genomes are AT rich (Aeschlimann et al., 2014; Aury et al.,
2006; Coyne et al., 2011; Eisen et al., 2006; Swart et al., 2013;
Wang et al., 2016). Reflecting their A/T mutational biases, among
eukaryotes with the highest UAA stop codon usage are standard
genetic code ciliates (Figures S7BS7D; Data S1V). This suggests that the diversification of genetic codes from the standard
one could have followed UAG and UGA stop codon depletion in
ancestral ciliates with AT rich genomes. While codon capture is a
reasonable explanation for the evolution of the Blepharisma genetic code (UAA stop codon usage 91%), it does not readily
explain the origin of other ciliate genetic codes. For example,
in Euplotes sp., according to tRNA anticodon-codon wobble
rules, UGG codons are expected to be misread as cysteine
following the origin of a tRNACys(UCA).
Even when relaxing the stop codon disappearance criterion (via
genetic code ambiguity tolerance), codon capture cannot easily
explain the general UAG and UAA reassignment trends seen in
Figure 1A. In all ciliates with reassigned UAG and UAA codons
and complete macronuclear genomes, both tRNAs with anticodon
complements of these codons are present (Aeschlimann et al.,
2014; Aury et al., 2006; Coyne et al., 2011; Eisen et al., 2006; Swart
et al., 2013). In the event that the first acquisition during codon reassignment was a tRNA(UUA), by the codon-anticodon wobble
rules UAA and UAG would both be translated; however, as this requires prior UAA stop codon disappearance, it is contrary to the
ciliate mutational tendencies. If codon reassignment were to occur
after a tRNA(CUA) acquisition, only UAG codons would be translated, and under the codon capture hypothesis, genetic codes
with UAG reassignment alone should be common; however, this
is not observed. Therefore, codon capture alone cannot explain
the diversity of genetic codes in ciliates.
As eRF1 recognizes stop codons, this protein could be a
determinant of genetic code reassignments in ciliates. Previously it was hypothesized that particular eRF1 amino acid substitutions are associated with each variant genetic code (Lozupone
et al., 2001). The additional ciliate genetic codes and eRF1 diversity present in ciliates and other eukaryotes present multiple
contradictions to the reported concordances between eRF1
amino acid substitutions and variant genetic codes (Lozupone
et al., 2001) (Figure S7A). Because no obvious associations between single eRF1 substitutions and variant genetic codes are
evident, any possible associations between genetic codes and
eRF1 changes must be more complex than individual amino
acid changes. The existence of the ambiguous ciliate genetic codes is also a challenge to explain by this hypothesis.
Because ciliate genetic code diversity does not seem to be
adequately explained by codon capture or eRF1 changes, we
instead propose that it is due to past genetic code ambiguity
tolerance and resolution, as exemplified by C. magnum and
Parduczia sp. Conversely, the inability to resolve ambiguity
favors the frozen state of the genetic code in other eukaryotes.
Cell 166, 691702, July 28, 2016 697
to substitutions that would cause premature translation termination in the standard genetic code. A potential drawback of such
robustness is that large insertions at 30 transcript ends may
expose stops that were previously translated. However, large insertions likely occur much less often than substitutions, and the
strong purifying selection governing non-protein-coding regions
in the heterotrich and karyorelict genomes will inhibit progressive
transcript end lengthening.
In summary, we propose that ambiguous ciliate genetic codes
are resolved by context-dependent translation termination, and
the reason why ciliates possess such diverse genetic codes is
that their ancestors had the ability to thrive for extended periods
with ambiguous genetic codes, as epitomized by C. magnum.
Together with the other variant genetic codes, these codes
show that the standard nuclear genetic code is not necessarily
an evolutionary dead end and that genetic codes can occasionally be observed in a state of flux. As highlighted here, the ambiguous genetic codes of C. magnum and Parduczia sp. also have
ramifications for our understanding of the suppression of translational readthrough, as well as how nonsense-mediated decay
(NMD) and selenocysteine translation operate (conserved proteins from both of these pathways are present in ciliates with
ambiguous genetic codes; see e.g., Figure S2E). To facilitate
future investigations concerning how sense is distinguished
from stop and related questions about codon disambiguation,
we have made a draft C. magnum macronuclear genome available under the accession number European Nucleotide Archive:
GCA_001499635.1.
EXPERIMENTAL PROCEDURES
See the Supplemental Experimental Procedures for additional detailed
protocols.
Transcriptomes Analyzed
Transcriptomes for C. magnum (MMETSP0210), Parduczia sp. (MMETSP1317),
and other eukaryotes assembled as part of MMETSP (Gentekaki et al., 2014;
Keeling et al., 2014)) were used to identify genetic codes and analyze stop codon
usage. We also predicted genetic codes after de novo assembling the transcriptomes of two peritrichous ciliates: Campanella umbellaria and Carchesium polypinum (NCBI short read archive: SRR1768423 and SRR1768437, respectively;
data from a recent phylogenomic study) (Feng et al., 2015) with Trinity (Grabherr
et al., 2011) (default parameters, version: trinityrnaseq_r20140717).
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AUTHOR CONTRIBUTIONS
Cosson, B., Couturier, A., Chabelskaya, S., Kiktev, D., Inge-Vechtomov, S.,
Philippe, M., and Zhouravleva, G. (2002). Poly(A)-binding protein acts in translation termination via eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation. Mol. Cell. Biol. 22, 33013315.
E.C.S. performed the computational analyses and assisted in laboratory experiments. V.S. cultured C. magnum, isolated nucleic acids and proteins,
and performed laboratory experiments searching for tRNAs. E.C.S. and V.S.
performed ribosome profiling. M.N. supervised the project. E.C.S. drafted
the manuscript with input from V.S., G.P., and M.N.
Coyne, R.S., Hannick, L., Shanmugam, D., Hostetler, J.B., Brami, D., Joardar,
V.S., Johnson, J., Radune, D., Singh, I., Badger, J.H., et al. (2011). Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living
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from the Mass Spectrometry and Proteomics Laboratory at the Childrens University Hospital in Bern for mass spectrometry support, Deis Haxholli for initial
genetic code inspections and the M.N. lab members for support and discussion. This research was supported by grants from the European Research
Council (ERC) (EPIGENOME) and National Center of Competence in Research
(NCCR) RNA and Disease to M.N., and the European COST Action BM1102.
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Article
Authors
Fatuel Tecuapetla, Xin Jin,
Susana Q. Lima, Rui M. Costa
Correspondence
fatuel@ifc.unam.mx (F.T.),
rui.costa@neuro.fchampalimaud.org
(R.M.C.)
In Brief
The direct and the indirect basal ganglia
pathway regulate movements by acting in
complementary supportive and
permissive manners, rather than by
having opposing hyperkinetic and
akinetic effects.
Highlights
d
Article
Complementary Contributions of Striatal Projection
Pathways to Action Initiation and Execution
Fatuel Tecuapetla,1,2,4,* Xin Jin,3 Susana Q. Lima,1 and Rui M. Costa1,4,*
1Champalimaud
Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida De Braslia, Lisbon 1400-038, Portugal
Molecular, Instituto de Fisiologa Celular, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria,
Circuito exterior s/n, Ciudad de Mexico 04510, Mexico
3Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
4Co-senior author
*Correspondence: fatuel@ifc.unam.mx (F.T.), rui.costa@neuro.fchampalimaud.org (R.M.C.)
http://dx.doi.org/10.1016/j.cell.2016.06.032
2Neuropatologia
SUMMARY
Lever press
Lever press start
Lever press end
Head entry
Lick
Sucrose 10 %
A
Task (FR8)
Day 1 of training
Magazine
ArchT expression
ChR2 expression
Str
Lever
Cx
Str
latency
Str
GP
Str
DAPI
RGS9L Cre ArchT-GFP
Day 11 of training
GP
GP
SNr
GPm
SNr
GPm
1 mm
50 sec
80
optogenetic
testing
session
60
40
20
0
CRF 1
11
Days in FR8
100
RGS9LArchT
RGS9LChR2
D1AchT
A2A/D2ArchT
D1ChR2
A2A/D2ChR2
80
60
40
20
0
CRF 1
11
Days in FR8
DAPI
D1 Cre ArchT-GFP
WT(C57BL/6J)
Str
Str
GPm
GP
SNr
GPm
DAPI
D1 Cre ChR2-eYFP
SNr
GPm
ArchT-GFP
NeuN
2
1
Str
CRF 1
11
DAPI
A2A Cre ArchT-GFP
Days in FR8
5
3
2
1
0
3
11
10 m
GP
DAPI
D2 Cre ChR2-eYFP
CRF 1
Str
GP
RGS9L Cre
40
60
D2/A2A Cre
D1 Cre
60
40
40
20
20
40
100
40
20
0
100
40
100
Days in FR8
RGS9L Cre
eYFP
Merged
RGS9L Cre
tdtom
Merged
Lateral
1.5 mm
100
Str
(%) positions
D1 Cre Opsin
D2/A2A Cre Opsin
100
80
BAC D1
tdtom RGS9L and D1 (+)
150 RGS9L (+)
BAC D2
eGFP RGS9L and D2 (+)
150 RGS9L (+)
100
50
10 m
10 m
0
1
Positions
# Cells
DAPI
# Cells
100
50
0
60
40
20
0
GP GPm SNr
Positions
Figure 1. Learning to Initiate and Perform Sequences of Lever Press in Cre Lines Expressing Opsins in the Different Striatal Projection
Pathways
(A) Representative example of the behavioral time stamps from one C57BL/6J mouse during two stages of training. Vertical lines represent the time stamps of
lever presses; they are color-coded to highlight the first and last press in a sequence.
(B) Proportion of sequences of presses (black) and individual presses (red) as training progressed for a group of C57BL/6J mice (upper) and for the different
cohorts of Cre lines expressing the opsins used in this study (bottom).
(C) Experimental setup showing the operant box and the position of an infrared beam in the path from the reward magazine to the lever.
(D) Mean latency between crossing the infrared beam and the first lever press of a sequence, as training progressed, for C57BL/6J mice (upper) and for the
different cohorts of Cre lines (bottom). The vertical arrows in (B) and (D) indicate when the optogenetic manipulations started.
(E) Representative sagittal mouse brain slices for the different Cre lines used in this study expressing either ArchT-GFP (left) or ChR2-eYFP (right). The reporter
signal (GFP or eYFP) is present in the striatum and in the nuclei that receive inputs from the infected striatal cells. Bottom middle: representative picture combining
three frames in the z axes (1 mm distance; confocal image) and depicting the presence of ArchT-GFP in the periphery of the labeled NeuN-positive cells.
(F) Quantification of expression of the opsins in different sections of each Cre line, using stereological quantification. The horizontal and vertical axes are the
percent of sections and the percent of NeuN-positive cells that also showed labeling for the opsin.
(G) To evaluate whether the RGS9L Cre line targets the two striatal pathways, we injected virus that expresses protein reporters into the striatum of RGS9L Cre
animals generated by crossing RGS9L Cre animals with either D1 td-tomato or D2-eGFP.
(H) Estimation of the proportion of D1 td-tomato-positive neurons from the neurons expressing eYFP after viral injection in RGS9L Cre.
(I) Estimation of the proportion of D2-eGFP-positive neurons from the neurons expressing td-tomato after viral injection in RGS9L Cre.
(J) Quantification of pixel intensity normalized to maximum intensity from three different target nuclei of the striatum. Cx, cortex; Str, striatum; GP, globus pallidus;
GPm, Globus pallidus medial and SNr, Subtantia nigra pars reticulata.
See also Figure S1 and Movie S1.
Magazine
RGS9L Cre
Lever
Lever
D1 Cre
D2/A2A Cre
latency
latency
infrared beam
triggers light
D1 Cre
(striatonigral-cells)
10
1
1
On
10
10
1
Lon
Seconds
10
On
Lon
(Light, 5 sec)
eYFP control
Lon
Ratio
ChR2-eYFP
10
Lon
Ratio
Light-on
eYFP control
On
10
ArchT-GFP
10
10
Lon
Ratio
10
Light-on
10
Light-on
10
10
On
On
**
10
Ratio
10
Lon
10
Lon
Ratio
3rd Block /2nd block
Seconds
0,3
L
Light-on
Light-on
10
On
1
10
On
10
10
Ratio
Light-on
Ratio
3rd Block /2nd block
Seconds
Lon
Light-on
10
Seconds
10
On
10
Seconds
(striatopallidal cells)
Ratio
Light-on
Seconds
10
10
Seconds
Seconds
Ratio
Light-on
11
Seconds
A2A/D2 Cre
3rd Block /2nd block
RGS9L Cre
(striatonigral-striatopallidal cells)
10
ChR2-eYFP
10
On
eYFP control
Lon
Figure 2. Optogenetic Modulations of the Activity of Striatal Projection Pathways before Sequence Initiation Increases the Latency to Initiate
the Action Sequence
(A) Experimental setup showing control blocks (light off, second block of a session) and optogenetic stimulation blocks (light on, third block of a session). To
investigate the contribution of the striatal pathway activity to the initiation of action sequences, light manipulations were triggered by crossing the infrared beam
when the animal moved from the magazine to the lever (red dot).
(B) Optic fiber placements for optogenetic manipulations in the DLS. Representative drawings depict the positions of the fiber tips from the animals used in this
study.
(C) Latency to initiate a sequence of lever presses for animals expressing either ArchT-GFP (dark green) or eYFP (light green) in both pathways (RGS9L); each
point is the mean latency for the session of one animal. Left: data from the last session without light manipulations (light off). Middle: a session with light manipulations (light on only during the third block). Right: ratios between the sessions with no light manipulations and those with light manipulation: Loff, off/off; Lon,
on/off.
(D and E) Same as in (C), except D1-Cre and D2/A2A-Cre animals were used, respectively.
(FH) Experiments presenting the effects of optogenetic activation of the striatal projection pathways with a 5-Hz stimulation on the latency to initiate the action
sequence (ChR2-eYFP, blue; eYFP, light green) for RGS9L, D1-Cre, or D2/A2A-Cre animals, respectively.
(IK) Same as in (F)(H), except, in this case, optogenetic activation occurred at 14 Hz;*p < 0.05; Wilcoxon test. Data are presented as the mean and SEM.
See also Figures S2 and S3 and Table S1.
activity before the first press of a sequence (using the D1Cre lines) also increased the latency to initiate the action
sequence (D1-Cre ArchT-GFP, light off = 4.5 1.0 versus light
on = 15.4 6.2; n = 9, Z = 2.66; p < 0.01, Wilcoxon test; Figure 2D,
middle). This effect remained consistent when controlling for any
potential order effect of the light-on and light-off blocks during
the session (n = 9; Z = 2.07; p < 0.05, Wilcoxon test; Figure 2D,
upper right; Table S1) and was not observed in D1-Cre animals
expressing only eYFP (n = 6, Z = 1.57; p > 0.05, Wilcoxon
test; Figure 2D, bottom right; Table S1). Interestingly, inhibition
of the activity of the striatopallidal pathway had a similar effect
in increasing the latency to initiate a sequence of lever presses
(2nd Block)
Magazine
Magazine
Lever
Lever
zone 1
switch
switch
zone 2
infrared beam
triggers light
# Switch Z1
ArchT-GFP
On
1
RGS9 Cre
0
Number of times
Off
5 sec
D2/A2A Cre
Top view
Cumulative frames
Tracking
Number of times
Off
On
Off
5 hz
5 sec
0
Off
On
0
Off
5 sec
On
14 hz
12
On
12
Number of times
D1 Cre
On
14 hz
5 hz
12
Off
Z2
ChR2-eYFP
On
Off
5 hz
On
14 hz
24
24
16
16
0
Off
On
Off
On
Off
On
Figure 3. Direct Pathway Manipulation Slows, while Indirect Pathway Manipulations Abort the Initiation of the Action Sequences
(A) Experimental setup with an arrow highlighting the possibility that animals would get out of the zone of performance of the task (Z1) during optogenetic
manipulations. Z1 was defined for each animal based on individual occupancy. See the Supplemental Experimental Procedures.
(BD) Left: gray pictures correspond to the upper view from three different animals in the behavioral box for each of the Cre lines depicted on the right (stimulation
at 14 Hz). Middle: heat color images of body occupancy from the videos acquired during the blocks of no light manipulation (off) versus the blocks of manipulation
(on). Color-code is normalized, where lighter colors show the areas in which animals spent more time during the corresponding block. Right: a representation of
the tracking of the center of mass for the same data. Light on, blue; light off, red.
(E) Quantification of the number of times animals left zone 1 for each manipulation. *p < 0.05, Wilcoxon test.
See also Figure S4 and Movies S2 and S3.
Light-off
(2nd Block)
Light-on
(3rd Block)
Magazine
Magazine
Lever
Lever
Z1
Z1
Z2
Z2
RGS9 Cre
D1 Cre
D2/A2A Cre
10
10
10
*
Off
Off On
On
*
Off On
On / Off
2
RGS9 Cre
D1 Cre
D2/A2A Cre
*
ArchT-GFP
eYFP control
# Switch Z1
Number of times
C
6
RGS9 Cre
Z2
D1 Cre
D2/A2A Cre
0
Off
On
0
Off
On
Off
On
lever press
5 hz L1
10
10
0
2
time (sec)
On
time (sec)
Light off
lever press
0
time (sec)
NS
On
Off
On
# Presses in sequence
Stim L4
# Lever in sequence
before vs after light
15
10
Off
15
NS
10
On
2nd
# Sequences
Sequences #
10
lever press
2
2
0
10
time (sec)
10
2nd
4th
# Presses during
stimulation
14 hz
14 hz
15
10
Seq
Seq per block
1
S-Lp
0
0
0
4th
14 hz
14 hz
K
Single Lp
10
NS
10
J
10
On
Latency to start
before vs after light
15
Light off
Off
5 hz L4
time (sec)
0
Off
F
# Lever presses
Sequences #
Light on (5 Hz L4)
12
On
5 hz L1
10
ChR2 eYFP
eYFP
E
12
Off
5 Hz L1
10
0
Off
# Lever presses
5 hz L1
15
Latency
to start
Seconds
# Lever presses
5 hz L1
15
# Presses during
stimulation
Seconds
# Presses in sequence
Stim L1
Stim L1 (early)
12
12
Sequences #
Light on (5 Hz L1)
S>2Lp
S Lp
S>2Lp
eYFP
eYFP
0
Off
On
Off
On
Off
On
Off
On
# Lever presses
Light off
# Lever presses
10
14 hz
0
Off
On
time (sec)
L
D1 Cre
Off
On
# Switch Z1
Number of times
Off
On
Top view
Cumulative frames
Z2
14 hz
Tracking
40
20
0
Off
On
Figure 5. Direct Pathway Activation during Sequence Execution Can Support Ongoing Action Performance
(A) Example of lever pressing from a D1-Cre mouse expressing ChR2-eYFP in the DLS direct pathway neurons during a no light block manipulation (left) and a light
block (5 Hz, 5 s, light activation was triggered by the first lever press of each sequence; Stim L1); note the increase in the number of lever presses during light stimulation.
(B) Left to right: quantification of the total number of lever presses for several animals subjected to the same manipulation as in (A). Middle (light green): control
experiments from animals expressing only eYFP that were subjected to the same protocol. Right: the total number of lever presses per sequence from a group of
animals subjected to stimulation immediately after the first lever press, except that the stimulation occurred early in training (early, during the first day of training in FR8).
(C) Mean number of lever presses during 5 s of light manipulation, 5 Hz, 5 s. In (A)(C), the first press in the sequence triggered the light activation.
(D) Latency to start a sequence of lever presses without (off) and with a 5-Hz stimulation triggered by the first press in a sequence.
(E) Example of a D1-Cre animal as in (A), except the light manipulation is triggered by the fourth lever press in the sequence (5 Hz, 5 s, Stim L4).
(F) Behavior from several animals as in (E).
(G) Number of presses per sequence before (second) versus a block after light manipulations (fourth; in this case, the animals were trained with four blocks).
(H) Comparison of the latency to start a sequence of lever presses before (second) and after light manipulation (fourth).
(I) Example of the lever-pressing behavior from an animal as in (A), except activation is at 14 Hz. In this case, instead of an increase in performance, there is a
pause in the sequence of lever presses during stimulation, and pressing immediately resumes after the light is turned off.
(J) Left to right: quantification of individual presses (Single-Lp) and sequences of lever press (Seq >, 2 Lp) for D1-Cre ChR2-eYFP or D1-Cre eYFP mice stimulated
at 14 Hz. The rightmost plot shows the quantification of the left panels presented as the ratio between the block of light on/light off.
(K) Mean number of lever presses during 5 s of light stimulation, 14 Hz.
(L) Number of exits from the performance zone (Z1) during the block of no light versus the block of a 14-Hz stimulation.
See also Figures S5, S6, and S7.
Light on (5 Hz L1)
lever press
# Lever presses
Sequence #
# Presses in sequence
Stim L1
10
10
0
0
time (sec)
5 hz L1
15
5 hz L1
10
Off
On
Off
5 hz L1
6
3
NS
On
NS
3
0
Off
14 hz
10
On
Latency
to start
Latency
to start
5 hz L1
time (sec)
# Presses during
stimulation
# Lever presses
Light off
Seconds
Seconds
Off
On
Off
On
ChR2-eYFP
eYFP
G
Light (14Hz)
2nd Block
3rd Block
Light off
4th Block
30
30
25
25
25
20
20
20
15
15
15
10
10
5
0
10
15
20
time (sec)
# Sequences
Sequence #
30
5
0
10
15
20
10
time (sec)
15
20
14 hz
14 hz
14 hz 15
14 hz
10
10
Single Lp
10
Seq
Seq per block
# Presses during
stimulation
# Lever presses
Light off
1
5
time (sec)
0
Off
On
Off
On
Off
On
Off
S Lp
S Lp
S>2Lp
eYFP
eYFP
S>2Lp
15
14 hz
10
0
Off
On
On
I
D2/A2A Cre
# Switch Z1
On
Off
1
Off
On
Cumulative frames
Tracking
Number of times
Top view
Z2
14 hz
40
20
0
Off
On
Figure 6. Increasing the Activity of the Striatopallidal Cells during the Execution of the Action Sequence Aborts Ongoing Performance
(A) Example of lever pressing for a D2/A2A-Cre animal expressing ChR2-eYFP in DLS during a no light block (light off, left) and a block of light stimulation triggered
by the first press (L1) in the sequence.
(B) Left to right: the total number of presses for several animals subjected to the same manipulation as in (A). Middle (light green): control experiments from animals
subjected to the same protocol, except the animals express eYFP.
(C) Mean number of lever presses during 5 s of light manipulation, 5 Hz. The first press in the sequence triggered light activation.
(D) Latency to start a sequence of lever presses for D2/A2A-Cre ChR2-eYFP animals during the block of stimulation at 5 Hz.
(E) Latency to start a sequence of lever presses for D2/A2A-Cre ChR2-eYFP animals during the block of stimulation at 14 Hz.
(F) Example of the performance of a D2/A2A-Cre ChR2-animal during no light blocks (left and right, light off) and the block with light stimulation at 14 Hz, triggered
by the first press in the sequence (L1) (light on, middle).
(G) Left to right: quantification of individual presses (single Lp) and sequences of lever press (Seq >, 2 Lp) from D2/A2A-Cre ChR2-eYFP or D2/A2A-Cre eYFP (light
on versus light off). Right: the quantification of the left, presented as the ratio between the blocks of light on/light off, is shown.
(H) Mean number of lever presses during 5 s of light manipulation, 14 Hz.
(I) Exits from the performance zone (Z1) during the block of light versus no light (14 Hz).
See also Figures S5, S6, and S7 and Movie S5.
ArchT-GFP
eYFP control
(Cont., 5 sec)
Light-on
12
8
4
RGS9 Cre
# Lever presses
ChR2-eYFP (5hz)
eYFP control
(5Hz, 5 sec)
(14Hz, 5 sec)
On
10
5
0
Lon Lon
Lon Lon
2
1
3
2
1
0
On
On
E
ChR2-eYFP (5hz)
eYFP control
(5Hz, 5 sec)
Light-on
10
5
0
On
On
15
10
5
0
1
0 L
3
15
10
5
0
On
L
on
on
2
1
0 L
On
Lon Lon
On
(14Hz, 5 sec)
# Lever presses
ChR2-eYFP (14hz)
eYFP control
0
On
Ratio
3
3rd Block /2nd block
Light-on
(Cont., 5 sec)
# Lever presses
ArchT-GFP
eYFP control
15
Lon Lon
15
Light-on
10
5
0
15
10
5
0
Lon Lon
15
Lon Lon
10
5
Lon Lon
2
1
0
On
3rd Block /2nd block
(14Hz, 5 sec)
# Lever presses
ChR2-eYFP (14hz)
eYFP control
On
On
3rd Block /2nd block
(5Hz, 5 sec)
# Lever presses
ChR2-eYFP (5hz)
eYFP control
Ratio
0
On
Light-on
(Cont., 5 sec)
# Lever presses
3
2
1
0
0
On
On
Lon Lon
# Lever presses
On
15
# Lever presses
ChR2-eYFP (14hz)
eYFP control
On
10
0
On
15
# Lever presses
Ratio
corresponding control animals for each Cre line are depicted. The panels on
the farthest right part of each row present the same data as in the three panels
explained above, except the data are normalized to the no light manipulation
block.
presses necessary to reach the same number of presses performed in sequences without stimulation, suggesting that areas
other than DLS can monitor and control the number of presses
that are supposed to be done in a sequence. This is even more
striking in the case, for example, of 14-Hz stimulation of the
dMSN were animals paused (Figures 5I and 5L), but resumed
performance immediately after pausing and completed the
same number of presses as in sequences without stimulation
(Figure 7F). Furthermore, with 5-Hz activation of the dMSN,
where animals increased the number of presses during stimulation (Figure 7E), they still performed more presses after stimulation, as if the presses driven by stimulation did not count for
the animal. Also, in the sequences that were not aborted during
14-Hz stimulation of the striatopallidal pathway (60%; Figure 6G),
animals performed sequences with the same number of presses
as with no light conditions (Figure 7I). These data suggest a
somewhat hierarchical organization of lever press sequences,
were the total length of elements in a sequence can be monitored
and encoded in circuits outside of these basal ganglia pathways.
However, that overactivation or inhibition of the striatopallidal
pathway (Figures 4C and 6F6I) was sufficient to abort
ongoing sequences and cause a switch in behavior, reveals
that sequence organization is more complex, and that changes
in activity of this pathway can abort an ongoing movement
sequence, and lead the animal to switch behaviors. Therefore,
these data support a mixed model of sequence organization,
with an overall hierarchical organization of action sequences,
but with striatopalidal neurons monitoring ongoing performance
and having the ability to abort the ongoing sequence and promote the switching to other behaviors.
Conclusions
In conclusion, the work presented here supports a model in
which both striatal projection pathways complement each other
during action initiation and performance, with the direct pathway
mainly supporting the selection/initiation and performance of
particular actions and the indirect pathway permitting proper
initiation by inhibiting competing movements or promoting
switching by monitoring ongoing performance and aborting
previous actions. These results show that appropriate complementary activity patterns in these pathways are critical for proper
motor control and may have important implications for pathological conditions that produce excessive repetitive behaviors or
excessive behavioral switching.
EXPERIMENTAL PROCEDURES
Animals
Male mice, 2- to 4-month-old, D1 Cre, A2A or D2 Cre and RGS9L Cre backcrossed into Black C57BL/6J or D1 td-tomato or D2 EGFP were used.
Training
We trained mice to develop self-paced sequences of lever press as previously
described (Jin and Costa, 2010; Jin et al., 2014). A sequence of lever presses
was defined as a bout of consecutive lever presses with no entry into the
magazine and licking.
Stereotaxic Virus Injections and Fiber Implantation
After anesthesia each animal was bilaterally injected using glass pipettes
with1 ml viral stock solution [DIO AAV2/1 ChR2-eYFP (UPENN) DIO AVV2/1
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Article
Authors
Juen Zhang, Lubin Tan, Yuqi Ren, ...,
Bernhard Bettler, Fengchao Wang,
Minmin Luo
Correspondence
luominmin@nibs.ac.cn
In Brief
Fear behavior in the face of danger relies
on presynaptic excitation in a habenular
circuit that is unexpectedly mediated by
GABAB receptors, synaptic proteins
typically responsible for inhibitory
signaling.
Highlights
d
Article
Presynaptic Excitation via GABAB Receptors
in Habenula Cholinergic Neurons
Regulates Fear Memory Expression
Juen Zhang,1,2,5 Lubin Tan,1,2,5 Yuqi Ren,2,3 Jingwen Liang,2 Rui Lin,2,3 Qiru Feng,2 Jingfeng Zhou,2,3 Fei Hu,2 Jing Ren,2
Chao Wei,2 Tao Yu,2 Yinghua Zhuang,2 Bernhard Bettler,4 Fengchao Wang,2 and Minmin Luo1,2,*
1School
SUMMARY
Fear behaviors are regulated by adaptive mechanisms that dampen their expression in the absence
of danger. By studying circuits and the molecular
mechanisms underlying this adaptive response, we
show that cholinergic neurons of the medial habenula reduce fear memory expression through GABAB
presynaptic excitation. Ablating these neurons or inactivating their GABAB receptors impairs fear extinction in mice, whereas activating the neurons or their
axonal GABAB receptors reduces conditioned fear.
Although considered exclusively inhibitory, here,
GABAB mediates excitation by amplifying presynaptic Ca2+ entry through Cav2.3 channels and potentiating co-release of glutamate, acetylcholine, and
neurokinin B to excite interpeduncular neurons. Activating the receptors for these neurotransmitters or
enhancing neurotransmission with a phosphodiesterase inhibitor reduces fear responses of both
wild-type and GABAB mutant mice. We identify the
role of an extra-amygdalar circuit and presynaptic
GABAB receptors in fear control, suggesting that
boosting neurotransmission in this pathway might
ameliorate some fear disorders.
INTRODUCTION
Excessive fear or fear that persists in the absence of threat is
dysfunctional, as in phobias and post-traumatic stress disorder
(PTSD) (Milad and Quirk, 2012; VanElzakker et al., 2014). The formation and expression of fear memory involves the extended
amygdala and its connected brain areas (Janak and Tye, 2015;
LeDoux, 2000; Tovote et al., 2015). The circuit between the amygdala and the limbic prefrontal cortex is critical for reducing fear responses during the extinction phase (Herry et al., 2010; Myers and
Davis, 2007). However, the functions and molecular mechanisms
of extra-amygdalar circuits in fear extinction remain unclear.
716 Cell 166, 716728, July 28, 2016 2016 Elsevier Inc.
AAV-flex-taCasp3-TEVp
taCasp3-2A-TEVp
EF1
taCasp3-2A-TEVp
WPRE
virus
EmGFP
overlay
EmGFP
Cre
EF1
ChAT
WPRE
100 m
taCasp3-TEVp
& EmGFP
conditioning
MHb
IPN
ChAT-Cre mouse
E
Freezing (%)
40
30
lesion (n=25) 60
ctrl (n=27)
day 2
day 3
****
****
lesion
ctrl
**
20
20
0
1 3 5 7 9
1 3 5 7 9
1 3 5 7 9
25
1 3 5 7 9
Trials
optical fiber
200 m
Freezing (%)
3 5
Test day
M
test day 1
day 2
day 3
60
IPN
Time (h
h20 s)
Test days
75
100 m
50
****
** 25
10
1 3 5 7 9
**
ctrl (n=9)
50
***
ctrl
lesion
75
lesion (n=13)
****
****
20
optical fiber
75
day 6
***
40
Trials
I
test day 1
lesion (n=12)
ctrl (n=18)
****
1 2 3 4 5
180 s
day 5
40
n.s.
day 4
60
****
test 2
G
test day 1
1s
20 s
test 1
F
conditioning day 0
50
test
20 s
conditioning
50
ctrl laser on
ChR2 laser off
ChR2 laser on
n.s. **
n.s.
40
n.s.
25
n.s.
20
****
****
0
1
Trials
Test day
Figure 1. Ablating Habenula Cholinergic Neurons Enhances the Expression of Conditioned Fear, whereas Activating These Cells Reduces
Fear Responses
(A and B) We generated a MHb-ChAT-taCasp3 (lesion) mouse or a MHb-ChAT-EmGFP (ctrl) mouse by infusing a mixture of AAV-DIO-EmGFP vectors and
AAV-flex-taCasp3-TEVp (A) or AAV-DIO-EmGFP only vectors into the MHb of a ChAT-Cre mouse (B).
(C) Coexpression of EmGFP (green) and ChAT (red) in the bilateral MHb of a control mouse (upper) and the lack of such expression and reduction of MHb volume
in a lesion mouse (lower).
(D) Methods for cued fear conditioning and tests. In test sessions, mice were exposed to either ten discrete conditioned stimuli (test 1) or one 180-s continuous
tone (test 2).
(E) Freezing response levels across trials during the cued fear conditioning on day 0.
(F) Freezing responses across trials in the extinction sessions on days 15, in which we presented ten discrete conditioned stimuli but omitted footshocks.
(G) Overall freezing responses of the lesion mice and control mice to discrete tones.
(H and I) Effects of lesion on the freezing responses (H) and overall freezing ratio (I) to a continuous 180 s tone.
(J) Distribution of ChR2-EYFP-expressing fibers (green) within the IPN and the method of delivering light with a tapered optical fiber.
(K) Method of testing the effect of optogenetic stimulation on freezing responses. Blue bar indicates light stimuli (5 ms light pulses at 50 Hz).
(L and M) Freezing responses across trials (L) and total freezing response levels (M) of the stimulation mice and control mice during extinction sessions. For a
control, we gave light stimulation to wild-type littermates or omitted light stimulation to ChAT-ChR2-EYFP mice.
*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant; two-way ANOVA on difference between phenotypes or between stimulation and control
(E, F, H, and L) and t tests (G, I, and M). Error bars indicate SEM.
See Table S1 for statistical analyses in detail. See also Figure S1.
phase (Figure 2E; Table S1), indicating that the GABAB activity of
cholinergic neurons is not required for forming cued fear memory. In the extinction sessions using discrete CS, a tone without
shock elicited significant higher freezing levels from GABAB CKO
mice, even after extinction training for 5 days (Figures 2F and 2G;
Table S1). When tested with a continuous 180-s tone, wild-type
mice gradually reduced their freezing, but the GABAB CKO mice
continued to freeze and exhibited significantly higher freezing
levels on days 1 and 6 (Figures S2J and S2K; Table S1). ChATGABAB(1)-KO mice did not show abnormality in locomotor activity before the tests, nor in general locomotion in an open field, nor
718 Cell 166, 716728, July 28, 2016
GABAB(1)
100 m
EYFP
EYFP
GABAB(1)
overlay
EYFP
100 m
EYFP
50
CKO (n=9)
G
test day 1
100
ctrl (n=9)
40
day 2
day 3
day 4
75
75
day 5
****
50
****
****
20
Trials
0
1
25
****
25
CKO
ctrl
**
50
ctrl (n=10)
****
n.s.
**
CKO (n=8)
30
10
overlay
IPN
overlay
IPN
GABAB(1)
MHb
100 m
GABAB(1)
100 m
MHb
Freezing (%)
overlay
Test day
Trials
Figure 2. Genetically Inactivating GABAB in Cholinergic Neurons Enhances the Expression of Learned Fear
(A and B) Strong GABAB(1) immunoreactivity (red) in habenula cholinergic neurons (green; A) and their axonal terminals in the IPN (green; B) of a ChAT-ChR2-EYFP
mouse.
(C and D) GABAB(1) immunoreactivity was markedly reduced in the MHb (C) and IPN (D) of a ChAT-GABAB(1)-KO (CKO) mouse. GABAB(1) expression remained
unchanged in the lateral IPN that received non-cholinergic inputs from the dorsal MHb.
(E) Freezing responses of both CKO mice and wild-type littermate controls (ctrl) during fear conditioning.
(F) Freezing responses of CKO mice and control mice during the fear extinction sessions on days 15. ****p < 0.0001 for difference between phenotypes in all
sessions, two-way ANOVA.
(G) Total freezing responses of the mutant and control mice. *p < 0.05; **p < 0.01; t tests. Error bars indicate SEM.
See Table S1 for statistical analyses in detail. See also Figure S2.
day 1
100
day 2
75
ACSF (n=6)
Saclofen (n=7)
****
75
day 3
Freezing (%)
Freezing (%)
***
50
****
25
50
n.s.
n.s.
25
0
1
Trials
100
Test day
100
ACSF (n=7)
baclofen (n=6)
75
Freezing (%)
Freezing (%)
ACSF
saclofen
****
50
****
25
ACSF
baclofen
75
**
50
25
**
****
0
0
1
Trials
100
n.s.
n.s.
75
ACSF (n=5)
baclofen (n=5)
Freezing (%)
Freezing (%)
n.s.
50
25
Test day
100
n.s.
ACSF
baclofen
75
n.s.
50
n.s.
25
0
0
1
Trials
2, baclofen 100 ms
ChAT-ChR2-EYFP
bac
2.0
2, baclofen
1.6
1.2
0.8
0.4
0
3, wash
1, ctrl
3.5
***
2.5
2.0
1.5
1.0
0.5
ctrl
balofen
10
15
20
25
30
35
baclofen
wash
GABA
puffing
100 pA
fr
50 ms
GABA
ChAT-ChR2-EYFP
wash
0.2
0.1
ChAT-ChR2-EYFP
& ChAT-GABAB(1)-KO
J
**
ctrl
0.8
0.6
fr
0.4
0.2
wash
20 ms
VGAT stimulation
0
ctrl
GABA
wash
ctrl
fr stimulation
1.0
**
n.s.
0
ctrl
200 pA
ctrl
10 ms
0.3
0
0
Time (min)
***
3.0
VGAT-ChR2-EYFP
500 pA
fr
3, wash
2.4
C
1, ctrl
20 pA
1.0
**
baclofen
***
0.8
0.6
0.4
0.2
0
Figure 4. Presynaptic GABAB Activity Drastically Potentiates Glutamate Release from Habenular Neurons
(AD) Baclofen (1 mM) produced an over tenfold increase in glutamatergic EPSCs. The cartoon in (A) illustrates the method of whole-cell recordings and
optogenetic stimulation of the ChR2-EYFP-expressing axons from the fasciculus retroflexus (fr). Raw traces (B) and a time-series plot of the EPSC amplitude (C)
draw data from one representative cell. The blue dot in (B) indicates 5-ms blue light stimulation. Horizontal lines in (C) show the timing of drug applications.
Glutamatergic EPSCs were isolated with the GABAA blocker picrotoxin (50 mM) and a mixture of nicotinic blockers hexamethonium (HMT, 50 mM) and mecamylamine (MEC, 5 mM). Numbers in (B) correspond to time in (C). n = 11 cells in (D).
(E and F) Raw traces from a single cell (E) and group data (F) show that knocking out GABAB(1) in cholinergic neurons abolished the potentiatory effect of baclofen
(n = 8 cells).
(GI) GABA puff (1 mM) potentiated glutamatergic EPSCs. Schematic in (G) illustrates the method of recording and GABA puff. Raw traces in (H) shows the
responses of a single cell and (I) shows group data (n = 7 cells).
(JL) Optogenetically stimulating (5 ms; 10 Hz, 15 s) GABAergic neuropils in the IPN of a VGAT-ChR2-EYFP mouse potentiated EPSCs that were evoked by
electric stimulation of the fiber tract fr. (J) illustrates the method of recording and optogenetic stimulation. (K) shows the raw traces from a single cell, with
stimulation artifacts clipped for clarity. (L) shows group data (n = 6 cells).
***p < 0.001; **p < 0.01; n.s., not significant; two-sided paired t tests. Error bars indicate SEM.
See Table S1 for statistical analyses in detail. See also Figure S4.
some cells (6/18 cells) expressed a large inward current that was
resistant to nicotinic blockers (Figure 5D). Consistent with the
expression of NKB in habenular neurons (Marksteiner et al.,
1992), the presence of NKB receptor antagonists abolished
this additional current in the six cells tested (Figures 5E and
5F; Table S1). This provides evidence both that NKB serves as
a neurotransmitter in the brain and that GABAB activity gates
the release of NKB.
Given that all three coreleased neurotransmitters evoked
excitatory currents, we tested whether baclofen could indeed
increase the ability of habenular neurons to elicit action potential
firing in interpeduncular neurons. We stimulated ChR2-expressing axonal terminals at various frequencies (5-ms pulses at
120 Hz for 1 s) and recorded action potential firing from interpeduncular neurons using cell-attached recording. Baclofen
significantly increased the number of action potentials evoked
by individual light pulses, especially when the stimulation frequency was <10 Hz (Figures 5G, 5H, S4R, and S4S; Table
S1). Therefore, presynaptic GABAB activity facilitates the spread
of excitatory signals across the habenulo-interpeduncular
synapse.
Next, we examined whether activating the receptors of
the coreleased neurotransmitters could reduce fear memory
expression. Wild-type C57BL6/N mice were conditioned with
200 pA
400 ms
2
1.0
baclofen (bac)
0.8
0.6
0.4
0.2
3
0
0
10
20
30
40
50
1.0
***
0.8
**
0.6
0.4
0.2
0
ctrl
bac
E
1
200 pA
400 ms
F
1.8
Time (min)
1.5
1.2
GR & SB
0.9
0.6
0.3
20
40
60
80 90
2.5
2.0
1.5
1.0
0.5
0
Time (min)
H
ctrl
baclofen
100
Evoked spikes/shot
***
3.0
***
Trial number
***
2.0
**
day1
Freezing (%)
100
day 2
day 3
100
Freezing (%)
ACSF (n=7)
AMPA&NMDA (n=6)
75
50
****
25
****
0
50
25
****
***
***
ACSF (n=7)
Senktide (n=6)
****
50
25
****
1
****
ACSF
Senktide
**
50
**
25
Test day
100
75
50
25
****
0
75
****
25
Test day
ACSF (n=7)
Bay 60-7550 (n=6)
50
n.s.
Figure 6. Activating Interpeduncular Neurons or Potentiating Habenular Neurotransmission Reduces Fear Memory Expression.
**
7
Trials
75
n.s.
25
100
****
1
*
50
75
ACSF
Acetylcholine
75
Freezing (%)
Freezing (%)
Trials
**
3
Test day
Freezing (%)
ACSF (n=7)
Acetylcholine (n=6)
100
Freezing (%)
***
2
100
100
25
75
***
Freezing (%)
Freezing (%)
50
100
75
****
Trials
ACSF
AMPA & NMDA
Trials
ACSF
Bay 60-7550
GCaMP6m
EF1
GCaMP6
AAV-EF1-DIO-GCaMP6m
ctrl
baclofen
wash
F/F
3
WPRE
2
Cre
EF1
GCaMP6m
WPRE
1
50 m
0.2
MHb
IPN
ctrl
baclofen
F/F
3
1
189
100 m
-3
-3
Time (s)
I
baclofen
F/F
J
100
F/F (%)
60
40
20
40 m
3
1
0.2
1.5
50 ms
-3
-3
Time (s)
baclofen
baclofen
baclofen
0.6
0.4
0.2
R
0
10
20
Time (s)
Ni2+
0.8
1.0
0.5
0.2
***
80
2.0
0.6
0.4
ctrl
baclofen
2.5
0.8
Time (s)
100 pA
100 m
1.0
F/F
ctrl
baclofen
F/F
30
40
50
1.2
***
***
***
0.9
0.6
0.3
0
60
Time (min)
overlay
IPN
CaV2.3
EYFP
overlay
2
1
50 ms
Q
0.6
baclofen
0.4
0.2
0
100 m
100 m
EYFP
CaV2.3
100 pA
10
15
20
25
0.4
n.s.
0.3
0.2
0.1
0
ctrl
baclofen
Time (min)
Figure 7. GABAB Mediates Excitation by Increasing Presynaptic Ca2+ Entry through Cav2.3-Containing Channels
(A and B) Injecting AAV-DIO-GCaMP6m vectors (A) into the MHb of a ChAT-Cre mouse resulted in GCaMP6 expression in MHb cholinergic neurons and their
axonal terminals in the IPN (B).
(CG) Baclofen (1 mM) increased GCaMP signals in the IPN. (C) Zoom-in view of GCaMP6 expression in the central IPN. (D) Pseudo-color representation of
GCaMP fluorescence change within the same area of (C) following electrical fiber stimulation (ten pulses at 10 Hz). (E) Raster plots of GCaMP6 signals for 189
terminal areas from three mice. (F and G) Average GCaMP signals in response to the single-shot (F) and trains (ten pulses at 10 Hz; G) of electrical stimulation.
Dashed lines indicate the timing of stimulation. Line width indicates SEM.
(H and I) Ni2+ blocked baclofens potentiatory effect on the evoked Ca2+ transients in the axonal terminals of habenula cholinergic neurons. (H) shows the
pseudocolor representation of GCaMP fluorescence changes within the IPN. (I) shows average amplitudes of Ca2+ transients evoked by single-shot electrical
stimulation.
(JL) Data from a single cell (J and K) and the entire test group (L) show that Ni2+ (50 mM) reversibly blocked EPSC potentiation by baclofen (n = 6 cells).
(M and N) Cav2.3 immunoreactivity was observed in ChR2-EYFP+ terminals within the IPN of a ChAT-ChR2-EYFP Ca2.3+/+ mouse (M), but not a Cav2.3 null
mouse (N).
(OQ) Data from a cell (O and P) and the entire test group (Q) demonstrate that mutating Cav2.3 prevented baclofen from potentiating EPSCs (n = 13 cells).
***p < 0.001; n.s., not significant; two-sided paired t tests in (I, L, and Q). Error bars indicate SEM.
See Table S1 for statistical analyses in detail. See also Figures S6 and S7.
responses to a previous danger-predicting cue. Our observations are consistent with the finding that inactivating or ablating
the fish homolog of the MHb causes helpless behaviors (Agetsuma et al., 2010; Lee et al., 2010), although it had been unclear
whether the fish effect could be extrapolated to mammals, as the
724 Cell 166, 716728, July 28, 2016
Because neither the MHb nor the IPN connects directly with
the amygdala, the current study raises the intriguing question
of how habenula neurons contribute to fear control. The habenulo-interpeduncular pathway connects the limbic forebrain
areas related to fear processing to several brainstem modulatory
centers, including the raphe nuclei (Figure S1A). Serotonergic
neurons in the dorsal raphe encode reward signals and selective
serotonin reuptake inhibitors alleviate the clinical symptoms
of PTSD (Li et al., 2016; Liu et al., 2014; Stein et al., 2000).
Possibly, habenula cholinergic neurons regulate the expression
of fear memory by controlling the activity of brainstem neurons,
including serotonergic neurons, thus modulating fear-related
forebrain areas, including the medial prefrontal cortex and amygdala (Herry et al., 2010; Myers and Davis, 2007).
GABAB Activity in Habenula Cholinergic Neurons
Facilitates Fear Extinction
A key finding of the present study is that GABAB activity on the
axonal terminals of habenula cholinergic neurons reduces
the expression of cued fear memory. Genetically inactivating
GABAB receptors in these neurons slows the decay of freezing
response levels during the extinction phase, but does not disrupt
fear memory formation or general locomotion, general anxiety, or
pain. Neurochemistry experiments further determine the IPN as
the key anatomical site for the fear-reducing effects of GABAB
activity. Blocking GABAB activity in the IPN immediately before
the extinction tests similarly increases freezing, whereas activating GABAB in the IPN reduces freezing in wild-type mice but
not in the GABAB conditional knockout mice. Tests using
discrete conditioned stimuli or 180-s continuous tone revealed
similar behavioral phenotypes, suggesting that the change in
fear responses is associated with memory expression rather
than sensory habituation.
Our results thus indicate that GABAB receptors in habenula
cholinergic neurons, particularly those in the axonal terminals,
are critically important for controlling responses to conditioned
stimuli that no longer predict threat. Pharmacological activation
of GABAB or the receptors for the coreleased neurotransmitters
in the IPN speeds up the decay, further supporting the assertion
that presynaptic GABAB activity facilitates fear extinction. On
the other hand, optogenetic stimulation of habenula cholinergic
neurons or infusion of a PDE2A inhibitor into the IPN reduces
the overall freezing response levels. Intra-IPN application of
saclofen increases the overall freezing response levels. Unlike
conditionally knocking out GABAB(1), infusing saclofen also inactivates GABAB receptors that are expressed by non-cholinergic
neurons within the IPN. Thus, the habenulo-interpeduncular
pathway can regulate both fear extinction and the overall fear
response intensity.
GABAB Mediates Presynaptic Excitation
Because GABAB activity had been thought previously to produce only inhibitory responses (Chalifoux and Carter, 2011;
Dutar and Nicoll, 1988a, 1988b; Gassmann and Bettler, 2012;
Newberry and Nicoll, 1984), it at first appears counterintuitive
that both the activity of habenula cholinergic neurons and the activity of GABAB receptors in their axonal terminals play similar
behavioral roles. Our finding that GABAB mediates strong pre-
CaV2.3 channels into precise microdomains in axonal terminals. The habenulo-interpeduncular pathway may serve as
a valuable model for dissecting the detailed molecular
mechanism underlying the rich signaling capacity of GABAB
receptors.
Taken together, this study reveals that GABAB receptors in
the habenula cholinergic neurons facilitate fear extinction by
potentiating corelease of multiple neurotransmitters. Malfunctions in the habenulo-interpeduncular pathway and in GABAB
signaling have been implicated in psychiatric disorders associated with fear and anxiety (Cryan and Kaupmann, 2005; Hikosaka, 2010; Lecourtier and Kelly, 2007). Our results suggest
that potentiating the habenulo-interpeduncular pathway, as
by activating habenular GABAB or inhibiting PDE2A, presents
a potentially effective therapeutic approach for treating such
disorders.
EXPERIMENTAL PROCEDURES
Detailed materials and methods are available in the Supplemental Experimental Procedures.
Mice
Adult mice of either sex were used. We used simplified genotypes
of mouse strains for clarity. Mouse strains include ChAT-ChR2-EYFP,
VGAT-ChR2-EYFP, ChAT-Cre, GABAB(1)lox511/lox511, Cav2.3 null, and wildtype C57BL6/N mice. All procedures were conducted with the approval
of the Animal Care and Use Committee of the National Institute of Biological Sciences, Beijing in accordance with governmental regulations of
China.
We thank G. Feng (Massachusetts Institute of Technology) for the ChATChR2-EYFP and VGAT-ChR2-EYFP transgenic mice, D. Duan (University of
Missouri) for advice on AAV virus preparation, Y. Li (Peking University) for
anti-synaptophysin1 antibody, and P. Sterling (University of Pennsylvania),
G. Marsicano (INSERM), and D. Perkel (University of Washington) for comments and discussions. M.L. is supported by China MOST (2012CB837701,
2012YQ03026005, 2013ZX0950910, 2015BAI08B02), NNSFC (91432114),
and the Beijing Municipal Government.
Received: November 8, 2015
Revised: March 2, 2016
Accepted: June 13, 2016
Published: July 14, 2016
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Theory
Authors
Mats Wallden, David Fange,
Ebba Gregorsson Lundius,
Ozden Baltekin, Johan Elf
Correspondence
johan.elf@icm.uu.se
In Brief
Cell-to-cell variation in division timing and
cell size in E. coli is due to differences in
growth rate, whereas the timing of
replication is triggered at an invariant
fixed volume per chromosome.
Highlights
d
Theory
The Synchronization of Replication
and Division Cycles in Individual E. coli Cells
Mats Wallden,1,2 David Fange,1,2 Ebba Gregorsson Lundius,1 Ozden Baltekin,1 and Johan Elf1,*
1Department
of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Husargatan 3, 75124 Uppsala, Sweden
author
*Correspondence: johan.elf@icm.uu.se
http://dx.doi.org/10.1016/j.cell.2016.06.052
2Co-first
SUMMARY
Isogenic E. coli cells growing in a constant environment display significant variability in growth rates,
division sizes, and generation times. The guiding
principle appears to be that each cell, during one
generation, adds a size increment that is uncorrelated to its birth size. Here, we investigate the mechanisms underlying this adder behavior by mapping
the chromosome replication cycle to the division
cycle of individual cells using fluorescence microscopy. We have found that initiation of chromosome
replication is triggered at a fixed volume per chromosome independent of a cells birth volume and
growth rate. Each initiation event is coupled to a division event after a growth-rate-dependent time. We
formalize our findings in a model showing that cellto-cell variation in division timing and cell size is
mainly driven by variations in growth rate. The model
also explains why fast-growing cells display adder
behavior and correctly predict deviations from the
adder behavior at slow growth.
INTRODUCTION
Balanced growth requires that DNA replication keeps up with
cell-division events (Schaechter et al., 1958). This may, at first,
seem hard to achieve for rapid-growth Escherichia coli, where
it takes more time to replicate a chromosome than to double
the biomass. The solution to the apparent paradox was first
described by Cooper and Helmstetter (Cooper and Helmstetter,
1968). In their model, new rounds of DNA replication are started
before the previous round has finished (Figure 1A). As long as
cells, on average, initiate one round of replication per chromosomal origin per generation and divide only once following
each replication termination, the model produces stable cell cycles at all growth rates. An example of a deterministic, i.e., noise
free, simulation of replication and division cycles including upand downshifts in growth rates is given in Figure 1B.
A missing component of Coopers and Helmstetters model
is how the cell manages to trigger replication initiation once
per generation. An answer proposed by Donachie (1968) is that
replication initiates at a critical volume per origin. This guarantees that, on average, the concentration of origins is constantly
Time
A
Cell volume (m3)
-1
-2
-1
-3
-2
-1
1
2
4
5
2
3
6
D
Cell volume (m3)
-1
-1
-3
2
1
2
4
-1
2
3
6
can therefore be seen as a reset point for the otherwise correlated variations in the cell cycle.
Time from Replication Initiation to Division Is GrowthRate Dependent
The next step in making a single-cell version of the CH model
was to determine how much time cells spend in replication,
segregating their chromosomes, and dividing (the C and the D
periods). Cooper and Helmstetter have assumed this time to
be constant for generation times faster than 60 min, and the
main question is thus if this holds at all growth conditions. By
mapping replication to the division cycle using the DnaQ data,
we determined the C and D periods for the different growth conditions (Figures 5A5C, Determining the C+D periods in DnaQYpet strain in the Supplemental Experimental Procedures). We
have found that the average C+D-period, t, is relatively constant
for the fast and intermediate growth conditions, but is much
longer at slow growth (Figure 5C).
Previous replication run-out experiments in bulk (Michelsen
et al., 2003) suggest a functional form for the growth-rate dependence as t = amb+g. In this equation, g can be thought of as the
minimal time for chromosome replication, chromosome segregation, and septum formation at fast-growth rates. Conversely,
at slow growth, these processes are limited by factors related
to growth, where a 1% decrease in growth rate results in approximately a b% increase in t . Fitting the data from the DnaQ strain,
which has been stratified based on growth rates in the different
media, gives a = 1.3, b = 0.84, and g = 42 (min). This implies
that t is strongly dependent on growth rate at slow growth.
To test if the t dependence estimated from the population averages applies also for individual cells, we used the data from the
SeqA-YFP strain, where initiation of replication could be accurately determined in individual cells (Figure 5D). Our result shows
two things. First, cells with the same growth rate in different media have different t and therefore t does not only depend on
growth rate. The difference in t for cells growing at the same
rate in different media in combination with a fixed initiation volume will result in difference cell sizes, which is in agreement
732 Cell 166, 729739, July 28, 2016
Time -->
1.5
0.5
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fit: 0.73/0.94
fit: 14/0.45
300
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0
0
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A
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Volume
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7
3
Cell Volume (m )
Model (variation in )
Experiment
7
Cell Volume (m )
0
1
Volume at Birth (m 3 )
100
200
300
Volume at Birth (m 3 )
Figure 6. The Growth Rate Variation Propagated to Other Variables and Compared to Experimental Observations
(A) The cell volume (black solid line) of a single cell lineage simulated according to the single-cell CH model throughout an upshift in growth conditions. The growth
rates are sampled from the observed distributions. When the cell passes the initiation volume(s) (red lines), a division event is triggered (green arrow) after a time
period corresponding to the C and D periods (gray bars). Variation can be introduced in growth rates, initiation volume, or C+D period.
(B) Top: The joint distribution of generation time and cell size at birth and division as observed in the DnaQ-YPet experiments. Bottom: The same data plotted as a
function of growth rate.
(C) Model predictions of the distribution in B when cell-to-cell variation is introduced in growth rate only. SD and means are as estimated from experiments
(Figure 2F). Here, SD/mean = 0.16, 0.23, and 0.23 were used for fast-, intermediate, and slow-growth rate, respectively.
(D) Same as (C), but cell-to-cell variation is also introduced into the initiation volume (SD/mean = 0.095) and C+D period (SD/mean = 0.05 for fast and intermediate
and SD/mean = 0.1 for slow growth).
(E) Marginal distributions for generation time and volume at birth from (C) shown as solid lines. Corresponding experimental distributions from (B) added for
comparison (dashed lines).
(F) Marginal distributions for generation time and volume at birth from (D) shown as solid lines. Corresponding experimental distributions from (B) added for
comparison (dashed lines). The cells included in experimental data are the same as in Figure 2.
See also Figures S5, S6, and S7.
C+D
Size
Added Volume (m )
Time
Size
Experiment
4
3
2
1
C+D
Model
1
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E
3
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3
Added Area(m2)
Birth Volume (m )
1.5
0.5
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2
Birth Area(m )
1.5
grown under fast conditions with 5 mM IPTG present in the medium to induce the
expression of FtsQ-GFP molecules. For the control experiment showing deviations from adder behavior (Figure 7E), strain JE201 was grown in slow-growth
conditions. Strain JE202 was studied under fast-growth conditions. All media
used in the microfluidic experiments contained a surfactant, Pluronic F108
(CAS 9003-11-6, Sigma-Aldrich), at a final concentration of 0.85 gLl1.
EXPERIMENTAL PROCEDURES
Strains
DnaQ localization was investigated using the MG1655 strain JJC5350 (a gift
from the lab of Benedict Michelle; Reyes-Lamothe et al., 2010), carrying a genetic fusion of the replication factor DnaQ and the yellow fluorescent protein
YPet, encoded in the native dnaQ locus. The construct was also transferred
to the MG1655 strain BW25993 (Datsenko and Wanner, 2000) using a P1
phage. We found the average growth rate in bulk experiments (Figures S1A
and S1B) and the growth-rate distribution and DnaQ localization (Figures S1I
and S1J) in microfluidics experiments to be very similar. Based on these similarities, we used the longest high-quality imaging data series regardless of
strain origin (JJC5350 in fast- and intermediate growth conditions and
BW25993 in slow-growth conditions) when comparing cell physiology to simulations (Figures 2, 6, and 7B). To increase the number of imaged cell cycles in
the fast- and intermediate growth conditions, we used both strains for DnaQ
localizations in Figures 3A3B.
SeqA localization (Figures 4, 5D, and 7D) was investigated using an MG1655
strain DS116 carrying a SeqA-YPet fusion. The strains were a kind gift from the
Waldminghaus lab, who replaced the native seqA gene with the seqA-ypet
fusion, constructed in the Radman lab (Babic et al., 2008).
The location of origins was investigated using strain JE200 (Figures 3D3F),
in which a genetic fusion of the transcription factor malI and the gene encoding
the yellow fluorescent protein variant Venus was introduced in the origin-proximal bgLG locus in strain BW25993 using the lambda-red protocol (Datsenko
and Wanner, 2000). The construct contains two tandem operator sites, malO,
to which MalI-Venus binds tightly. Further, the native malI gene, as well as
the native malO sites, was deleted using the lambda-red method. This minimal
construct was selected to avoid the risks associated with having a large number
of operator-transcription factor complexes present in the cell (Lau et al., 2003).
Precision in the determination of division timing (Figure S2; see Validation
of the phase contrast division classifier the Supplemental Experimental Procedures ) was investigated in strain EC442 (Soderstrom et al., 2014), containing a genetic fusion of the division factor, ftsQ, and a green fluorescent protein,
gfp, introduced in MG1655.
Accuracy in determining individual growth rates (Figure S3D; see Comparing
growth rates in phase contrast and fluorescence in the Supplemental Experimental Procedures) and the control experiment for deviation from adder
behavior (Figure 7E) was investigated in strain JE201, in which a gene encoding
a red fluorescence protein, tRFP, regulated by the constitutive ribosomal RNA
promoter P2rrnB was introduced at the intC locus using the lambda-red method
in BW25993.
The dependence of the growth rate on ribosome content (Figure S7; see
Dependence of growth rate and ribosome content and partitioning at birth
in the Supplemental Experimental Procedures) was studied in strain JE202,
in which gene rpsB was genetically fused to yellow fluorescence protein,
Venus, and gene rplI to a red fluorescent protein, mCherry, by using the
lamda-red protocol. In both cases the constructs replaced the native genes.
The rpsB and rplI genes express the proteins S2 and L9 that associate to
the small and large subunit of the ribosome, respectively.
Growth Conditions
Growth conditions are as follows: fast: M9 minimal medium and 0.4% glucose
supplemented with RPMI 1640 amino acids (R7131, Sigma-Aldrich) at
37 C; intermediate: M9 minimal medium and 0.4% succinate supplemented
with RPMI 1640 amino acids (R7131, Sigma-Aldrich) at 30 C; and slow:
M9 minimal medium and 0.4% acetate at 37 C.
All strains were grown in M9 minimal media, except for the experiment that determines the accuracy in individual growth rates (Figure S3D), in which the strain
JE201 was grown in Luria-Bertani liquid medium (LB) at 37 C. Strain EC442 was
phase-contrast image were segmented using the method described in Sadanandan et al. (2014). An active contour model based on Sliusarenko et al.
(2011) was developed, and a contour was computed for each segmented object. Cells were tracked between frames using the method described in Magnusson et al. (2015). The output was filtered so that each cell cycle must have a
parent and two children and that the maximum displacement of that cell between any consecutive frames was less than the cell width. Growth-condition-dependent filter criteria were added so that only cells with a cycle time
of 50, 30, and 15 min were retained for slow, intermediate, and fast growth,
respectively. To attain high-precision volume estimates, the estimated volumes from the active contours were fitted to V(t) = VB exp(mt), where t is the
time from cell birth, VB is the birth volume, and m is the growth rate. Cell generations were filtered based on root-mean-squared errors of the fit being
smaller than 5% of the average cell volume. The fitted volumes at times of division and initiation were used in all figures, except for Figure 3, where a large
number of cell generations were required and it is sufficient to know the size of
the cell regardless of division parameters. The analysis of the precision in division timing is described in the Supplemental Experimental Procedures (see
Validation of the phase contrast division classifier) (Figure S2). The determination of length, areas, volumes, and widths was based on the contour model
as in Sliusarenko et al. (2011).
Phase-contrast and fluorescent images of strain JE202 and the fluorescent
images of JE201 were segmented and tracked as described above, but not
fitted with active contours.
Replisomes and origins were detected in the raw fluorescence images using
the method described in Hammar et al. (2014). The coordinates were set in a
cellular reference frame using the contour model derived from the phasecontrast image taken in conjunction with the fluorescence image.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and two movies and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.06.052.
AUTHOR CONTRIBUTIONS
M.W., J.E., and D.F. designed the experiments. D.F., J.E., and M.W. developed the model. J.E., D.F., and M.W. wrote the paper; M.W., D.F., E.G.L.,
and O.B. made the experiments; M.W. and D.F. made the analysis codes
and performed analysis; and D.F. built the setup and made the simulation
code and the simulations.
ACKNOWLEDGMENTS
We thank Gustaf Ullman, Alexis Boucharin, Erik Marklund, and Prune Leroy for
valuable support in programming, data analysis, and cloning; Sajith Kecheril
and Carolina Whalby for providing image analysis code; Bill Soderstrom for
providing EC442; Benedict Michelle for providing JJC5350; and Torsten Waldminghaus and Daniel Schindler for providing DS116. This work was supported
by the European Research Council, Vetenskapsradet, and the Knut and Alice
Wallenberg Foundation.
Received: December 11, 2015
Revised: March 25, 2016
Accepted: June 28, 2016
Published: July 28, 2016
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Resource
Authors
Hui Zhang, Tao Liu, Zhen Zhang, ...,
Daniel W. Chan, Karin D. Rodland,
the CPTAC Investigators
Correspondence
dchan@jhmi.edu (D.W.C.),
karin.rodland@pnnl.gov (K.D.R.)
In Brief
Layering proteomic and genomic data
from ovarian tumors provides insights
into how signaling pathways correspond
to specific genome rearrangements and
points to the benefit of using protein
signatures for assessing prognosis and
treatment stratification.
Highlights
d
Resource
Integrated Proteogenomic Characterization of
Human High-Grade Serous Ovarian Cancer
Hui Zhang,1,15 Tao Liu,2,15 Zhen Zhang,1,15 Samuel H. Payne,2,15 Bai Zhang,1 Jason E. McDermott,2 Jian-Ying Zhou,1
Vladislav A. Petyuk,2 Li Chen,1 Debjit Ray,2 Shisheng Sun,1 Feng Yang,2 Lijun Chen,1 Jing Wang,3 Punit Shah,1
Seong Won Cha,4 Paul Aiyetan,1 Sunghee Woo,4 Yuan Tian,1 Marina A. Gritsenko,2 Therese R. Clauss,2 Caitlin Choi,1
Matthew E. Monroe,2 Stefani Thomas,1 Song Nie,2 Chaochao Wu,2 Ronald J. Moore,2 Kun-Hsing Yu,5,6 David L. Tabb,3
David Fenyo,7 Vineet Bafna,8 Yue Wang,9 Henry Rodriguez,10 Emily S. Boja,10 Tara Hiltke,10 Robert C. Rivers,10
Lori Sokoll,1 Heng Zhu,1 Ie-Ming Shih,11 Leslie Cope,12 Akhilesh Pandey,13 Bing Zhang,3 Michael P. Snyder,6
Douglas A. Levine,14 Richard D. Smith,2 Daniel W. Chan,1,16,* Karin D. Rodland,2,16,* and the CPTAC Investigators
1Department
SUMMARY
INTRODUCTION
To provide a detailed analysis of the molecular components and underlying mechanisms associated
with ovarian cancer, we performed a comprehensive
mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by
The Cancer Genome Atlas (TCGA), of which 169
were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the
genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated
with chromosomal instability, the sets of signaling
pathways that diverse genome rearrangements
converge on, and the ones most associated with
short overall survival. Specific protein acetylations
associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable
resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational
modification levels and clinical outcomes in HGSC.
A comprehensive molecular view of cancer is necessary for understanding the underlying mechanisms of disease, improving
prognosis, and ultimately guiding treatment (Hanahan and Weinberg, 2011). The Cancer Genome Atlas (TCGA) conducted an
extensive genomic and transcriptomic characterization of
ovarian high-grade serous carcinoma (HGSC) aimed at defining
the genomic landscape and aiding the development of targeted
therapies for this highly lethal malignancy (Cancer Genome Atlas
Research Network, 2011). Key findings from TCGA were: (1) the
prevalent role of TP53 mutations, (2) extensive DNA copy alterations, (3) preliminary transcriptional signatures associated
with survival, (4) varied mechanisms of BRCA1/2 inactivation,
and lastly, (5) CCNE1 aberrations. Subsequent analysis of
genomic data from the TCGA consortium led to the refinement
of the transcript-defined signatures, improving the statistical
association with patient outcome (Yang et al., 2013), and integrating microRNA and mRNA expression profiles associated
with HGSC to identify candidate microRNA targets (Creighton
et al., 2012).
While the insights from genomic analyses are substantial,
functions encoded in the genome are generally executed at the
protein level and are often further modulated by post-translational modifications (PTMs) (Vogel and Marcotte, 2012). For
example, TCGA used a reverse-phase protein array (RPPA)
Cell 166, 755765, July 28, 2016 2016 Elsevier Inc. 755
3.0
Median = 0.45
1.0
1.5
2.0
0.0
0.5
Probability Density
2.5
-1.0
-0.5
0.0
Spearmans Correlation
0.5
1.0
Protein
mRNA
DNA replication
Protein
cell-cell communications
differentiated
metabolic
cytokine signaling
proliferative
mesenchymal
stromal
ECM interaction
mRNA
differentiated
complement cascade
immunoreactive
proliferative
mesenchymal
n.a.
metabolism
Z-score
Four of the proteomic clusters showed a clear correspondence to the mesenchymal, proliferative, immunoreactive, and
differentiated subtypes defined by the TCGA transcriptome analysis (Figure 2; Figure S2E). A relatively small fifth cluster of
tumorssignificantly enriched in proteins associated with extracellular matrix interactions, erythrocyte and platelet functions,
and the complement cascadewas also observed using
multiple approaches, including model-based clustering with
Bayesian information criteria, consensus clustering, and Visual
Statistical Data Analyzer (VISDA)-based sub-phenotype clustering. This new group could not be attributed to tissue source
site sampling bias or any other metadata category but may be
related to tumor characteristics, including vascularization and
tumor content, as the tumor purity score for this subtype (and
the mesenchymal subtype) was significantly lower than that of
the other three subtypes (Figure S2F). The clinical relevance of
these protein-based clusters will require validation in independent HGSC sample sets, particularly as no significant difference
in survival was observed (Figure S2G), similar to mRNA-based
clustering analysis (Cancer Genome Atlas Research Network,
2011).
Proteomic Analysis of CNA Effects
Chromosomal instability, marked by extensive copy-number alterations (CNAs) in each tumor, is a hallmark of HGSC and a likely
source of driver alterations in this disease (Cope et al., 2013; Kuo
et al., 2009; Kobel et al., 2008). CNAs can affect the abundance
758 Cell 166, 755765, July 28, 2016
of proteins at the same locus (cis effects) and may also act in
trans either directly or indirectly.
Hypothesizing that CNAs with strong trans effects are more
likely to elicit a molecular phenotype and confer selective advantages, we sought to identify those CNA regions that have the
broadest effect on protein expression. In all, 29,393 CNA loci
(Table S5) were compared to our global proteomics data, with
950,209 CNA/protein pairs (0.72% of the total) exhibiting significant association (Benjamini-Hochberg adjusted p value < 0.01).
We provide a complete list of the significantly associated proteins for each CNA in Table S5. The diagonal line evident in Figure 3 corresponds to cis effects, and vertical stripes correspond
to trans effects, in which changes in copy number affect expression of numerous proteins across the genome. We performed
the same analysis for mRNA to identify sites where associations
are transcriptionally mediated. A similar number of CNA/mRNA
pairs were found to be significantly associated (1,113,164 at a
Benjamini-Hochberg adjusted p value < 0.01; Figure 3). This
analysis revealed regions on chromosomes 2, 7, 20, and 22
correlated in trans with more than 200 proteins. In contrast to
colorectal cancer, where most of the trans-regulation of protein
by CNA was accompanied by similar changes in mRNA (Zhang
et al., 2014), we observed several loci associated with differences in protein abundance without a corresponding change in
mRNA. For example, large regions on chromosome 2 have relatively little trans effect on mRNA levels but are associated with
more than 200 proteins in trans. Dissecting the mechanisms by
CNA-protein correlation
6
4
3
2
1
100 200 300 400
-200 -100 0
Significant
correlations in trans
Gene location
15 17 19 21
7 8 9 10 11 12 13 14 16 18 20 22 X Y
CNA-mRNA correlation
8 9 10 11 12 1314 16 18 20 22 X Y
15 17 19 21
CNA location
8 9 10 11 12 1314 16 18 20 22 X Y
15 17 19 21
CNA location
1.00
0.75
Signature
relative level
0.50
p value = 2e-6
Up
Down
0.25
0.00
0
4
Time (years)
Figure 6. DDN Analysis and Lysine-Acetylation Analysis between HRD and Non-HRD
Patients
SWATH data
p value = 0.028
HRD negative
HRD positive
HRD status
Histone H4 GLGK(Ac)GGAK(Ac)R
(peak area)
500000 1000000 1500000
Histone H4 GLGK(Ac)GGAK(Ac)R
(log ratio)
-1.0 -0.5 0.0 0.5 1.0 1.5
p value = 0.039
HRD negative
HRD positive
HRD status
A
0
10
20
15
44.8
RhoA regulatory
31.0
PDGFR
24.4
Integrin-liked kinase
Notch
HER2/Neu
Rac1
Cxcr4
Thrombin
IL-12
Phosphopeptide
Protein
Transcript
CNA
Thrombaxane
m = mRNA
P = protein abundance
P = phosphoprotein
= increased (0.1>p>0.05)
= increased (p<0.05)
= decreased (0.1>p>0.05)
= decreased (p<0.05)
= no difference
Nucleus
parsimonious Cox proportional hazards models with maximal predictive ability, using the PNNL data for training and JHU data for testing. Kaplan-Meier
plots were used to visualize performance in predicting overall survival and progression-free survival. A CIN index (Cancer Genome Atlas Research Network,
2012) was calculated for each sample as the mean absolute values of copynumber measurements at the 29,393 selected loci. Bootstrap resampling
was used to select proteins correlated with CIN index at high confidence.
Protein Acetylation and HRD
Acetylated peptides were identified by searching the global proteomics data
for dynamic acetylation to lysines (+42 Da). Acetylation levels were compared
between HRD and non-HRD cases by t test. Targeted proteomics (SWATH;
Collins et al., 2013) was used to orthogonally quantify the acetylated peptide
with synthetic peptides as the internal standard.
Survival-Related Pathways Analysis
Phosphoproteome data from the short and long survivors were mapped to
signaling pathways in the NCI Pathway Information Database (PID) (http://
pid.nci.nih.gov) using the gene names. For each signaling pathway in the
PID, relative abundances for all phosphopeptides mapping to any pathway
component were identified and separated into short- and long-survivor
groups. The difference in distributions between the two sets of pathway-specific peptides, i.e., those associated with either short survivors or long survivors, was then assessed using a two-tailed t test. Similar enrichment analyses
were also performed using protein abundance, mRNA abundance, and CNA.
Data Repository
All of the primary MS data on TCGA tumor samples are deposited at the
CPTAC Data Coordinating Center as raw and mzML files and complete
protein assembly datasets for public access (https://cptac-data-portal.
georgetown.edu).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.05.069.
A video abstract is available at http://dx.doi.org/10.1016/j.cell.2016.05.
069#mmc10.
CONSORTIA
The members of the National Cancer Institute Clinical Proteomics Tumor Analysis Consortium are Steven A. Carr, Michael A. Gillette, Karl R. Klauser, Eric
Kuhn, D.R. Mani, Philipp Mertins, Karen A. Ketchum, Ratna Thangudu, Shuang
Cai, Mauricio Oberti, Amanda G. Paulovich, Jeffrey R. Whiteaker, Nathan J.
Edwards, Peter B. McGarvey, Subha Madhavan, Pei Wang, Daniel Chan, Akhilesh Pandey, Ie-Ming Shih, Hui Zhang, Zhen Zhang, Heng Zhu, Leslie Cope,
Gordon A. Whiteley, Steven J. Skates, Forest M. White, Douglas A. Levine,
Emily S. Boja, Christopher R. Kinsinger, Tara Hiltke, Mehdi Mesri, Robert C.
Rivers, Henry Rodriguez, Kenna M. Shaw, Stephen E. Stein, David Fenyo,
Tao Liu, Jason E. McDermott, Samuel H. Payne, Karin D. Rodland, Richard
D. Smith, Paul Rudnick, Michael Snyder, Yingming Zhao, Xian Chen, David
F. Ransohoff, Andrew N. Hoofnagle, Daniel C. Liebler, Melinda E. Sanders,
Zhiao Shi, Robbert J.C. Slebos, David L. Tabb, Bing Zhang, Lisa J. Zimmerman, Yue Wang, Sherri R. Davies, Li Ding, Matthew J.C. Ellis, and R. Reid
Townsend.
AUTHOR CONTRIBUTIONS
Study Conception & Design, H. Zhang., T.L., Z.Z., R.D.S., D.W.C., and K.D.R;
Investigation Performed Experiment or Data Collection, T.L., S.S., F.Y.,
J.-Y.Z., Lijun Chen, P.S., P.A., Y.T., M.A.G., T.R.C., C.C., S.T., S.N., R.J.M.,
H. Zhang, and H. Zhu. Computation & Statistical Analysis, Z.Z., S.H.P., Bai
Zhang, J.E.M., V.A.P., Li Chen, D.R., M.E.M., S.W.C., S.W., J.W., D.L.T.,
D.F., V.B., Y.W., and Bing Zhang. Data Interpretation & Biological Analysis,
T.L., H. Zhang, S.H.P., Z.Z., Bai Zhang, J.E.M., VA.P., Li Chen, D.R., S.N.,
C.W., I.-M.S., A.P., M.P.S., D.A.L., R.D.S., D.W.C., and K.D.R. Writing Manuscript Preparation & Revision, H. Zhang, T.L., Z.Z., S.H.P., J.E.M., D.R., V.A.P.,
L. Cope, H.R., I.-M.S., A.P., M.P.S., D.A.L., R.D.S., D.W.C., and K.D.R. Supervision & Administration: H.R., E.S.B., T.H., R.C.R., L.S., R.D.S., D.W.C., and
K.D.R.
ACKNOWLEDGMENTS
This work was supported by National Cancer Institute (NCI) CPTAC awards
U24CA160019 and U24CA160036 and by NIH grant P41GM103493. The
PNNL proteomics work described herein was performed in the Environmental
Molecular Sciences Laboratory, a U.S. Department of Energy (DOE) National
Scientific User Facility located at PNNL in Richland, WA. PNNL is a multi-program national laboratory operated by the Battelle Memorial Institute for the
DOE under contract DE-AC05-76RL01830. Genomics data for this study
were generated by the TCGA Pilot Project, established by the NCI and the National Human Genome Research Institute.
Received: December 21, 2015
Revised: April 5, 2016
Accepted: May 19, 2016
Published: June 29, 2016
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Resource
Authors
Ulrike Kusebauch, David S. Campbell,
Eric W. Deutsch, ..., Leroy Hood,
Ruedi Aebersold, Robert L. Moritz
Correspondence
aebersold@imsb.biol.ethz.ch (R.A.),
rmoritz@systemsbiology.org (R.L.M.)
In Brief
This resource enables the accurate
detection and quantification of any
known or predicted human protein from
complex biological samples.
Highlights
d
Accession Numbers
GSE83654
Resource
Human SRMAtlas: A Resource of Targeted Assays to
Quantify the Complete Human Proteome
Ulrike Kusebauch,1 David S. Campbell,1 Eric W. Deutsch,1 Caroline S. Chu,1 Douglas A. Spicer,1 Mi-Youn Brusniak,1
Joseph Slagel,1 Zhi Sun,1 Jeffrey Stevens,1 Barbara Grimes,1 David Shteynberg,1 Michael R. Hoopmann,1
Peter Blattmann,2 Alexander V. Ratushny,1,6 Oliver Rinner,2,3 Paola Picotti,2 Christine Carapito,2 Chung-Ying Huang,1
Meghan Kapousouz,1 Henry Lam,4 Tommy Tran,1 Emek Demir,5 John D. Aitchison,1,6 Chris Sander,5 Leroy Hood,1
Ruedi Aebersold,2,7,* and Robert L. Moritz1,*
1Institute
SUMMARY
20,277 proteins
PABST
evaluate sequence characteristics
rank observed & predicted peptides
final suitability score
observed
peptides
empirical suitability score
PeptideSieve,
32,418 SNPs
N-glycosylated proteins
A S
P R
intensity
MS/MS
6530 Q-TOF
data directed
5 CEs
CE
www.srmatlas.org
www.peptideatlas.org
www.proteinatlas.org
www.nextprot.org
www.pathwaycommons.org
www.srmcollider.org
the detection of a transition a full MS/MS spectrum was acquired to create a QTrap spectral library. SRM assay parameters including precursor and fragment ion
type, charge state, and rank order, elution time as well as chromatograms, MS/MS spectra, and CE plots are provided in the human SRMAtlas resource. The
human SRMAtlas is integrated with external knowledge bases providing comprehensive information on a protein of interest.
See also Figure S1 and Table S2.
166,174
149,265
peptides
160,000
126,712
158,015
149,961
120,000
80,000
40,000
0
selected
6530
Q-TOF
6460
QQQ
5500
combined
QTRAP
B 100
C 100
80
80
60
60
40
40
20
20
Success rate in %
Selected peptides in %
0
6 8 10 12 14 16 18 20 22 24 26 28 30
Peptide length
D 100
E 100
80
80
60
60
40
40
20
20
3
4
5
6
Expected charge state
0
5 10 15 20 25 30 35 40 45 50 55 60
A C D E F GH I L MN P Q S T VWY
SSRCalc
Amino acid
F 100
G 100
80
80
60
60
40
40
20
20
0
A C D E F G H I K L MN P Q R S T VWY
N-terminal amino acid
A C D E F G H I K L MN P Q R S T VWY
C-terminal amino acid
20,000
proteins
15,000
10,000
5,000
0
0
2
3
4
5+
any
peptides
selected peptides
all developed assays
6530 Q-TOF assays
QTrap 5500 assays
6460 QQQ assays
charge state (z) of 2 (61.3%), 3 (28.5%), and 4 (6.3%) were preferably selected and performed generally better compared to a
small number of peptides that fragmented with z = 1 (C-terminal
peptides) or z R 5 (long peptides with several basic residues).
Further, we found that peptides with an SSRCalc value of seven
to 46 performed best and that cysteine containing peptides
showed a decreased success rate compared to all other peptide
sequences (Figures 3B3G; Figure S2).
Next, we reassessed the protein coverage achieved by the
158,015 successfully developed SRM assays, taking into account that some peptides failed to result in the correct synthesis
product or to fragment with sufficient quality. The generated assays covered 99.7% of the predicted human proteome with at
least one SRM assay per protein and provide a minimum of three
assays for 19,337 (95.4%) of all UniProtKB/Swiss-Prot annotated human proteins (Figure 4). We were able to develop a minimum of four assays for 91.5% and at least five assays for 85.3%
of the proteome, respectively. Taken together, on average, each
protein of the human proteome is represented by eight SRM
assays per protein and some by more than 25 peptides. The
assessment of the SRM assay chromatographic performance
utilizing the 6460 QQQs was as successful. For 98.9% of the predicted human proteome, we were able to acquire high-quality
SRM traces with at least one peptide per protein, while 90.3%
of the proteome is represented by three SRM assays.
During the course of the project, updated versions of the
UniProtKB/Swiss-Prot database were released. To account for
new protein entries, we developed 443 additional SRM assays
for 162 entries and included these assays in our database to
provide SRM assays for updated human reference proteomes
2014 (20,193 proteins) and 2015 (20,203 proteins) (Figure S3;
Table S4). Overall, we have successfully developed 158,015
mass spectrometric assays based on high-quality MS/MS
spectra and subsequent QQQ deployment with the use of
166,174 chemically synthesized peptides to reliably identify
772 Cell 166, 766778, July 28, 2016
Huh7
citrate
ACLY
acetyl-CoA
II
III
SREBP2
IV
22.2
6x
untreated
25
y10 - 1130.5324+
y7 - 805.3686+
b3 - 348.1918+
y9 - 1015.5055+
y6 - 706.3002+
b9 - 1026.4415+
22.2
LPDS +
5M atorvastatin
15
10
40.4
untreated
22.2
18
19 20
21
22 23
24
25
18
19 20
21
22 23 24
25
40.4
LPDS +
5M atorvastatin
3
2
1
0
y9 - 1015.5055+
y6 - 706.3002+
b9 - 1026.4415+
8x
10
y10 - 1130.5324+
y7 - 805.3686+
b3 - 348.1918+
CYP51A1
LBR
TM7SF2
MSMO1
NSDHL
HSD17B7
EBP
SC5D
Vitamin D
DHCR7
bile acids, steroids
y9 - 1100.5881+
y8 - 494.2557++
b4 - 457.2405+
20
DHCR24
FDFT1 - LFSASEFEDPLVGEDTER
y10 - 1214.6310+
y8 - 987.5040+
b3 - 344.1565+
20
15
Dolichol, Ubiquinone
HepG2
y9 - 1100.5881+
y8 - 494.2557++
b4 - 457.2405+
25
atorvastatin
DHCR24 cholesterol
Intensity
y10 - 1214.6310+
y8 - 987.5040+
b3 - 344.1565+
ACAT2
HMGCS1
HMGCR
MVK
PMVK
MVD
IDI1
FDPS
FDFT1
SQLE
LSS
CYP51A1
LBR
TM7SF2
MSMO1
NSDHL
HSD17B7
EBP
SC5D
DHCR7
response-based cluster
protein
LPDS + 5M atorvastatin
FDFT1 - TQNLPNC[160]QLISR
LPDS + 1M atorvastatin
0.01% DMSO
untreated
LPDS + 5M atorvastatin
LPDS + 1M atorvastatin
log2FC
untreated
3 2 1 0 1 2 3
0.01% DMSO
CYP51A1
MSMO1
FADS2
HMGCS1
NSDHL
LSS
NEU1
DHCR24
NUCB2
FDFT1
DHCR7
HMGCR
IDI1
MVD
ACLY
FDPS
MVK
ACAT2
EBP
PLIN2
HSPA5
FKBP11
FAM117A
SC5D
AGR2
HIST1H2*
ACTB/G*
NDRG1
LBR
SEC24D
IDH1
FASN
GAPDH*
PGM3
TUBA1A
GFPT1
40.4
37
38 39 40 41 42 43 44
1
0
37 38 39 40 41 42 43 44
Retention Time
Figure 5. Drug-Induced Inhibition of Cholesterol Synthesis
Systematic proteomic quantification of proteins from a reported gene module and enzymes in the cholesterol synthesis pathway upon drug treatment.
(A) The heatmap shows the change in protein abundance following the treatment with lipoprotein-deficient serum (LPDS) and atorvastatin compared to control
conditions (untreated and 0.01% DMSO) of the same cell line. The signal represents the mean result from three independent biological experiments and three
SRM analyses per sample. Proteins were hierarchically clustered according to the elicited response with the Ward2 algorithm and Euclidean distance. Based on
the clustering tree and the protein regulation, we defined five different clusters of proteins showing similar response (IV). Proteins marked with asterisks were
included as housekeeping proteins for normalization.
(B) Shown are the measured proteins from the pathway synthesizing cholesterol from acetyl-CoA. The enzymes are sorted by their position in the pathway, and
the proteins are color coded according to the cluster in (A) they belong to (I)(IV). The proteins or metabolites in italics have not been measured and are included for
completeness. The inhibition of HMGCR by atorvastatin and the negative feedback of SREBP2 are depicted.
(C) SRM chromatograms of peptide TQNLPNC[160]QLISR and LFSASEFEDPLVGEDTER from protein FDTF1 showing the difference in signal abundance between untreated cells and cells treated with LPDS + 5 mM atorvastatin as representative examples. The lower signal in untreated cells is magnified in the inset.
See also Table S5.
plasma membrane
cytoplasm
nucleus
8h
24 h
48 h
72 h
PRIM1
1.5
RNA
SRM
RNA
LNCAP SRM
RNA
PC3
SRM
DU145
0
-1.5
Figure 6. Network of Proteins Associated with Docetaxel Perturbation of the Cell Cycle
SRM-based quantification of a protein network in three prostate cancer cell lines, DU145, LNCaP, and PC-3, at four time points post-treatment with docetaxel
and in untreated controls in comparison to mRNA abundance changes. The microarray-derived functional network was visualized with ingenuity pathway
analysis (IPA). The structure of the network is based on the IPA Core Analysis-, STRING-, and Pathway Commons-derived direct interactions and indirect relationships. Each heatmap visualizes the log2 abundance change of treated versus control cells for each time in each cell line at the transcript (mRNA) and protein
(SRM) level. The signal represents the mean result from two technical replicates at the transcript level and three SRM analyses per sample.
See also Figure S4 and Table S5.
mine the state of a network and to compare network states between samples, the parts of a network need to be consistently
detected in sample cohorts and quantified, at least at the level
of relative quantification. An incomplete parts list may not explain
observed processes. The Human SRMAtlas assays provide the
tools to reliably navigate predicted or experimentally derived
protein networks, to rapidly probe promising interaction partners
and to perform relative or, in the presence of isotope-labeled
reference peptides, absolute quantification and to thus provide
new insight into complex biological mechanisms.
The developed assays can be universally applied in different
sample types to target any protein of interest and its abundance
changes. The assays are beneficial in hypothesis-driven experiments focusing on a relatively small number of proteins, e.g.,
Cell 166, 766778, July 28, 2016 775
therefore, optimized protein extraction, enrichment, and fractionation can further improve sensitivity.
While we present a truly comprehensive and highly characterized resource of assays for all human proteins, their variants and
some post-translational modifications, the human proteome is
complex, and the coverage by empirical observed peptides is
evolving and by no means complete. We provide very high
coverage (>99%) at the protein level, but the protein sequence
coverage by peptides, in their native and post-translational modified form, will increase as more data become available. Our established pipeline will allow us to develop SRM assays for newly
identified peptides and different post-translational modifications
in future efforts with ease. The human SRMAtlas is based on the
UniProtKB/Swiss-Prot database 2010 and 2015, but, as the
proteome continues to be refined, new assays can be added.
The human SRMAtlas not only facilitates the reliable identification of proteins, but also provides coordinates for the reproducible quantification of analytes. Absolute quantification of
peptides can be accomplished by adding known amounts of stable isotope-labeled standards. Label-free quantification can also
be performed by SRM but is only accurate for relative quantification given there is no standardization anchor point. Alternatively,
absolute label-free quantification based on few anchor points
can be pursued (Ludwig et al., 2012). Currently, the resource
does not provide experimentally determined limits of detection
(LOD) and limits of quantification (LOQ) for every SRM assay
as these parameters strongly depend on the individual setting
in which the assay is deployed (e.g., cell lysate versus plasma,
type of sample preparation, chromatographic performance of
LC system). In order to obtain the most accurate LOD/LOQ,
these values need to be determined in the individual sample
type in the context of each study. We introduced PASSEL (Farrah
et al., 2012) as a repository for SRM data to share deployed
assays and their performance in different matrices and similar
databases followed (Sharma et al., 2014; Whiteaker et al.,
2014). While SRM assays developed in different laboratories
may be available as part of a publication or through these repositories, the information is scattered, time-consuming to gather,
and currently limited to a small number of proteins (<1,000). In
contrast, the human SRMAtlas provides high-quality SRM assays including their spectral libraries for the entire predicted human proteome developed in a consistent manner. These assays
are generic and are not based on any sample type or biological
context but have proved to work in a number of settings (cell
lines, plasma, urine protein digests, etc.).
The SRM assays presented here also provide a unique
resource for efforts seeking to provide protein-level evidence of
any human protein either previously observed or never observed
to date to advance our knowledge in human biology and complex diseases. The high-resolution, high-mass accuracy MS/
MS spectra generated from synthetic peptides constitute a
gold-standard fragmentation database that can provide additional confidence by spectral comparison with MS/MS spectra
derived from discovery proteomic studies. Recently, the concept
of SWATH-MS was introduced (Gillet et al., 2012), a data-independent MS technique that is less sensitive than SRM but
capable of generating a digital map of a large fraction of a proteome. The analysis of SWATH data requires information from
AUTHOR CONTRIBUTIONS
R.L.M. and R.A. conceived the project. U.K., D.S.C., E.W.D., P.B., O.R., P.P.,
and R.L.M. designed and interpreted experiments. U.K., D.S.C., E.W.D.,
C.S.C., D.A.S., M.-Y.B., J. Slagel, Z.S., J. Stevens, B.G., D.S., M.R.H., P.B.,
A.V.R., C.C., C.-Y.H., M.K., and T.T. performed experiments and bioinformatics calculations. D.S.C., E.W.D., H.L., U.K., E.D., and C.S. carried out database design, integration, and deployment. U.K., D.S.C., R.A., and R.L.M.
wrote the manuscript. J.D.A., L.H., and all authors contributed to the preparation of the manuscript.
ACKNOWLEDGMENTS
This work was performed in part with federal funds from the American Recovery and Reinvestment Act (ARRA) funds through NIH, from the National
Human Genome Research Institute grant RC2HG005805 (to R.L.M.), the National Institute of General Medical Sciences under grant R01GM087221,
S10RR027584 and 2P50 GM076547/Center for Systems Biology (to
R.L.M.), the Luxembourg Centre for Systems Biomedicine/University
Luxembourg (to L.H.), the European Research Council grant ERC-2008AdG 233226 and ERC-2014-AdG 670821 and the Swiss National Science
Foundation (grant #31003A-130530) (to R.A.), and DAAD (fellowship to
U.K.). We kindly thank K. Miller and C. Miller (Agilent Technologies), J. Louette, Drs. G. Sulyok and A. Schierholt (Thermo-Fisher), Dr. H. Wenschuh and
L. Eckler (JPT) for their support, Dr. S. Carr for early access to ESP predictor,
Drs. P. Gaudet and A. Bairoch for supporting the integration of neXtProt, and
T. Farrah, S-T. Kwok, A. Aksoy, and P. Shannon for excellent technical
support.
Received: December 8, 2015
Revised: April 28, 2016
Accepted: June 21, 2016
Published: July 21, 2016
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Editorial Note
Systemic Spread of Sequence-Specific Transgene RNA
Degradation in Plants Is Initiated by Localized
Introduction of Ectopic Promoterless DNA
Olivier Voinnet, Philippe Vain, Susan Angell, and David C. Baulcombe*
*Corresponding author
http://dx.doi.org/10.1016/j.cell.2016.07.014
779
Editorial Note
A Viral Movement Protein Prevents Spread of the
Gene Silencing Signal in Nicotiana benthamiana
Olivier Voinnet, Carsten Lederer, and David C. Baulcombe*
*Corresponding author
http://dx.doi.org/10.1016/j.cell.2016.07.015
Editorial Note
Ordered Recruitment of Transcription
and Chromatin Remodeling Factors to a Cell Cycle
and Developmentally Regulated Promoter
Maria Pia Cosma, Tomoyuki Tanaka, and Kim Nasmyth*
*Corresponding author
http://dx.doi.org/10.1016/j.cell.2016.07.016
781
Editorial Note
LINE-1 Activity in Facultative
Heterochromatin Formation during
X Chromosome Inactivation
Jennifer C. Chow, Constance Ciaudo, Melissa J. Fazzari, Nathan Mise, Nicolas Servant, Jacob L. Glass, Matthew Attreed,
Philip Avner, Anton Wutz, Emmanuel Barillot, John M. Greally, Olivier Voinnet, and Edith Heard*
*Corresponding author
http://dx.doi.org/10.1016/j.cell.2016.07.013
782 Cell 166, 782, July 28, 2016 2016 Elsevier Inc.
784
DOI http://dx.doi.org/10.1016/j.cell.2016.07.022
autophagy
rllS: dFOXO,
reduced DILP production
Neuroendocrine
AMPK
Metabolic
homeostasis
Gustatory inputs
Or 83b Gr 63a
X X
Olfactory receptors:
Sensory perception
DILPs
Drosophila
CRTC-1
Cytoplasm
AMPK
aboli h
eos as
Metabolic
homeostasis
IL Ps
Longevity
Insulin signaling
Autophagy
Fat deposition
Stress resistance
Serotonin synthesis
Reduced
educed IIS (rIIS):
rIIS
Age-1/daf-2 mutations
Neuroendocrine
eu oe docrine
Proteostasis
UPR ER (XBP-1s)
Chronic stress
HSF-1
Mitochondrial stress
Nutrient availability
Dietary restriction
SKN-1
Nutrient type
NMUR-1
HIF-1
Low O 2
Olfactory and gustatory
inputs
Octopamine
Sensory perception
Sensor
erce tion (extrinsic)
extrinsic
Neuronal mechanisms
DAF-16, HSF-1
CRTC1
NF- B
GnRH
IKK /
rllS: Irs2/IGF-1R
Immune inhibition:
Neuroendocrine
FMO-2
ox2r
Neural activation
SIRT1
Neurogenesis,
cognitive improvement
CGRP
Longevity
Longevity
Youthful mitochondrial
function and morphology
Pancreas
Glucose homeostasis
Respiration
Mitochondrial metabolism
Mitochondrial dynamics
Mouse
DAF-16
activity
Stress resistance
Pro-longevity gene transcription
UPR ER, UPR mt
Respiration
espirat on
Xenobiotic detoxification
Metabolic
homeostasis
TR PV1
Cytoplasm
Cellular Exogenous
stress
Hsp70
Pain
Sensory perception
UPR
C. elegans
784.e1
DOI http://dx.doi.org/10.1016/j.cell.2016.07.022