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Centrosome duplication is tightly controlled during faithful Recent studies indicate that cellular components that regulate nucleo-
cell division, and unnecessary reduplication can lead to cytoplasmic transport are independently involved in spindle assembly9,10.
supernumerary centrosomes and multipolar spindles that are In principle, these processes are accomplished by specific receptors of
associated with most human cancer cells 1–5. In addition to the β-importin family: for example, the importin receptors (importins
nucleocytoplasmic transport, the Ran–Crm1 network is involved α and β) that bind to nuclear localization signals (NLS) and the export
in regulating centrosome duplication to ensure the formation receptor Crm1 that binds to nuclear export signals (NES). These proc-
of a bipolar spindle6–8. Here, we discover that nucleophosmin esses require a small GTPase, Ran, which controls the interaction of
(NPM) may be a Ran–Crm1 substrate that controls centrosome these receptors with their substrates. The guanine nucleotide-exchange
duplication. NPM contains a functional nuclear export signal factor RCC1 facilitates Ran binding to Crm1, whereas RanBP1, a major
(NES) that is responsible for both its nucleocytoplasmic regulator of Ran, promotes Crm1 dissociation from Ran. Importins α
shuttling and its association with centrosomes, which are and β may negatively regulate NLS-containing proteins to modulate
Ran–Crm1-dependent as they are sensitive to Crm1-specific microtubule assembly. We have recently shown that Crm1 may regu-
nuclear export inhibition, either by leptomycin B (LMB) or by late the fidelity of centrosome duplication by acting as a licencing fac-
the expression of a Ran-binding protein, RanBP1. Notably, tor to prevent unscheduled duplication6. A fraction of Ran, Crm1 and
LMB treatment induces premature centrosome duplication RanBP1 is found on centrosomes6,7,11. Inactivation of Crm1, either by
in quiescent cells, which coincides with NPM dissociation a Crm1-specific inhibitor, LMB12, or through hepatitis B virus (HBV)
from centrosomes. Moreover, deficiency of NPM by RNA HBx protein interaction with its NES motif13, results in supernumerary
interference results in supernumerary centrosomes, which can centrosomes and multipolar spindles6,13. Similar multipolar spindles
be reversed by reintroducing wild-type but not NES-mutated and mitotic abnormalities are also a consequence of Ran mutations or
NPM. Mutation of a potential proline-dependent kinase overexpression of RanBP1 (ref. 8). We hypothesize that the Ran–Crm1
phosphorylation site at residue 95, from threonine to aspartic complex may negatively regulate the initiation of centrosome duplica-
acid (T95D) within the NES motif, abolishes NPM association tion, possibly through its association with NES-containing proteins6.
and inhibition of centrosome duplication. Our results are NPM may negatively regulate centrosome duplication14,15. NPM is a
consistent with the hypothesis that the Ran–Crm1 complex ubiquitously expressed phosphoprotein that is mainly localized in the
may promote a local enrichment of NPM on centrosomes, nucleolus, and shuttles between the nucleus and the cytoplasm during
thereby preventing centrosome reduplication. the cell cycle16. NPM associates with unduplicated centrosomes and dis-
sociates from centrosomes upon phosphorylation by CDK2–cyclin E,
The centrosome is the principal microtubule-organizing centre of ani- which coincides with the initiation of centrosome duplication and DNA
mal cells, and functions in concert with the spindle poles to direct the replication15,17. NPM reassociates with centrosomes during mitosis15,18.
assembly of a bipolar spindle during mitosis1,2. Centrosome duplication Sequence analysis of NPM orthologues indicated the presence of a puta-
is initiated at the G1/S boundary and is completed at the S phase of the tive hydrophobic leucine-rich NES motif (IxxPxxLxL) within residues 94–
cell cycle, which coincides with DNA replication. To ensure the forma- 102 that is conserved from Xenopus laevis to human (Fig. 1a). A putative
tion of a bipolar mother cell, duplication takes place only once per cell bipartite NLS motif and nucleolus localization signal (NoLS) are also evi-
cycle, leading to an equal division into two daughter cells1,2. This feature dent (Fig. 1a). The presence of both NES and NLS motifs in NPM suggests
indicates that there are negative regulatory proteins that act as licencing that a Ran GTPase-dependent pathway regulates NPM nucleocytoplasmic
factors to prevent unscheduled duplication. However, the precise mecha- transport. Moreover, it is possible that the Ran–Crm1 complex modulates
nism of how centrosome duplication is controlled is largely unknown. centrosome duplication by regulating NPM transport and localization.
1
Liver Carcinogenesis Section, Laboratory of Human Carcinogenesis, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA.
2
Correspondence should be addressed to X.W.W. (e-mail: xw3u@nih.gov)
GFP−NPM-positive
80
m-NPM KLDEDDEDDD EDDEDD-EDD DDDDFDEEET EEKVPVKKSV RDTPAKNAQK SNQ 208
human nuclei
r-NPM KLDEDDDEDD EDDEDD-EDD DDDDFDEEET EEKVPVKKSV RDTPAKNAQK SNQ 208
ch-NPM KLSEDDEDDD EDEDDD--ED DEDDLDDDEE EIKTPMKKPA REPAGKNMQK AKQ 208 60
xl-NPM RLEEEEEDSD EDDDDD--DE DDDDEDDDEE EEETPVKK-T DSTKSKAAQK LNH 207
Consensus .L.......D .D..DD.... D.DD.D..E. E...P.KK.. .....K..QK ...
40
CDC2 CDC2
h-NPM NGKDSKPSST PRS-KGQESF KKQE-K--TP KTPKGPSSVE DIKAKMQASI EKG 258
m-NPM NGKDLKP-ST PRS-KGQESF KKQE-K--TP KTPKGPSSVE DIKAKMQASI EKG 256 20
r-NPM NGKDLKP-ST PRS-KGQESF KKQE-K--TP KTPKGPSSVE DIKAKMQASI EKG 256
ch-NPM NGKDSKP-ST PAS-KTKTPD SKKD-KSLTP KTPKVPLSLE EIKAKMQASV DKG 258
xl-NPM NGKASALSTT QKTPKTPEQK GKQDTKPQTP KTPKTPLSSE EIKAKMQTYL EKG 260
0
Consensus NGK......T ....K..... .K...K..TP KTPK.P.S.E .IKAKMQ... .KG Control LMB
NoLS
h-NPM GSLPKVEAKF INYVKNCFRM TDQEAIQDLW QWRKSL 294
m-NPM GSLPKVEAKF INYVKNCFRM TDQEAIQDLW QWRKSL 292
r-NPM GSLPKVEAKF INYVKNCFRM TDQEAIQDLW QWRKSL 292
ch-NPM CSLPKLEPKF ANYVKNCFRT EDQKVIQALW QWRQTL 294
xl-NPM NVLPKVEVKF ANYVKNCFRT ENQKVIEDLW KWRQSLKDGK 300
Consensus ..LPK.E.KF .NYVKNCFR. ..Q..I..LW .WR..L....
d e
NLS (141−157) WT NESM NESD NLS1D
NES (94−102) NoLS (288−290)
WT GFP - 294
A-A
NESM GFP -
NESD GFP -
NLS1D GFP -
NLS2D GFP -
G-G
NoLSM GFP -
A-A NLS2D NoLSM NESM-NLS1D NLS1/2D
NESM-NLS1D GFP -
NLS1/2D GFP -
Figure 1 Nucleocytoplasmic shuttling of NPM depends on the presence of cells, identified under phase contrast (phase), GFP–NPM signal, human
functional NES and NLS motifs mediated by the Ran network. C23 signal, as well as their merged images, are shown. Magnification:
(a) Sequence comparison of NPMs from five different species was performed ×400. (c) Percentage of heterokaryon fusion cells with the GFP–NPM signal
by the ClustalW program. The NES (yellow), NLS (sky blue) and nucleolus present in human nuclei was quantified by counting more than 100 fusion
localization signal (NoLS; green) motifs are highlighted. Potential CDC2 cells. Values were obtained from three independent experiments, shown
phosphorylation sites (red) and a proline-dependent kinase (PDK) site as mean ± s.d. (d) Schematic depiction of GFP–NPM and its NES, NLS
(red) were determined by Motif Scan. NPM fusion protein sites (grey) that and NoLS mutants. WT, wild type; NESM, mutated at residues L100A and
result from translocation of the N-terminal NPM to ALK, RAR, or MLF1 L102A; NESD, deleted at residues 94–102; NLS1D, deleted at residues
in lymphoma and leukaemia are also indicated. Prefixes: h, human; m, 134–142; NLS2D, deleted at residues 150–155; NoLSM, mutated at
mouse; r, rat; ch, Chinese hamster; xl, Xenopus laevis. (b) The ability of residues W288G and W290G; NESM-NLS1D, with both a L100A/L102A
NPM to shuttle from the nucleus to the cytoplasm was determined by the mutation and a 134–142 deletion; NLS1/2D, deleted at residues 134–142
mouse-human heterokaryon assay in the absence (upper panels) or presence and 150–155. (e) Intracellular localization of the NPM mutants. HCA2-
(lower panels) of the nuclear export inhibitor leptomycin B (LMB). Human hTERT cells were transfected with various mutants, incubated for 24 h, fixed
nucleolin-specific antibody C23 (red) was used to identify human nuclei with paraformaldehyde, and examined under a fluorescence microscope.
(arrows) from mouse nuclei in the heterokaryon. Representative fusion Representative images are shown. Magnification: ×400.
We tested this hypothesis by using a heterokaryon assay in which a antibody was used to distinguish human cells from murine cells (Fig. 1b;
mouse–human cell fusion was induced by polyethylene glycol19,20 to evalu- arrows). The green fluorescent protein (GFP)–NPM signal, predomi-
ate whether NPM in mouse nuclei was exported to the cytoplasm, and then nantly localized in the nucleolus, was frequently found not only in murine
transported into human nuclei. A human-specific anti-nucleolin (C23) nuclei, but also in human nuclei where they were a part of a heterokaryon
a b Endogenous NPM c
NPM γ-tubulin Merge GFP−NPM
GFP−NPM-NESM
Cells with
75
Cells with
G0/G1
60
50
40
25
20
0
0
MB
P1
l
tro
G0/G1 S/G2 M
HB
nB
n
+L
Co
Ra
S/G2
d e
n ≥ 2 centrosomes (%)
100 n=1
Cells with n = 1 or
n≥2 G1: 91% G1: 91%
75 S: 4% S: 3%
G2/M: 5% G2/M: 6%
M 50
25
0
− LMB + LMB
− LMB + LMB
f
Control 50 nM LMB
NPM
100
40
Mitotic cells with
Centrosomes
20 10
0
0
B
l
tro
LM
B
l
tro
n
LM
Merge
Co
n
nM
Co
nM
50
50
Figure 2 The centrosome association of NPM and initiation of centrosome of 40 nM LMB for 2 h, or with expression of HBx or GFP–RanBP1. More
duplication are sensitive to Ran–Crm1-specific nuclear export inhibition. than 100 cells were analysed. Data are an average of three independent
(a) Localization of NPM on G0/G1 phase, S/G2 phase, or mitotic phase experiments and are shown as mean ± s.d. (d) The percentage of cells
centrosomes. HCA2-hTERT cells were fixed and stained with anti-NPM that contain either one (n = 1) or two or more (n ≥ 2) centrosomes was
(green panels) and anti-γ-tubulin for centrosomes (red panels). The following calculated in serum-starved HCA2-hTERT cells treated with or without LMB.
representative cells with different stages of centrosomes (arrows) and their At least 100 cells were counted for each experiment, and data are shown as
merged images from NPM and γ-tubulin staining are shown: normal cells mean ± s.d. of three separate experiments. (e) Cell-cycle profiles of serum-
with a single centrosome representing the G0/G1 phase; an interphase starved cells in d were determined by FACS analysis. (f) Effect of LMB on
cell with two adjacent centrosomes representing the S/G2 phase; and binding of NPM to mitotic centrosomes. Mitotic HCA2-hTERT cells were
a mitotic cell with two distanced centrosomes representing M phase. collected by shaking and were reseeded in chamber slides in the absence or
Magnification: ×630. (b) The percentage of interphase cells from serum- presence of 50 nM LMB for 2 h. Representative images from control or LMB-
starved HCA2-hTERT cells with NPM-bound centrosomes or mitotic cells treated mitotic cells, as analysed by anti-NPM (green) or anti-γ-tubulin (red)
from unsynchronized cells with or without the expression of GFP–NPM or antibodies are shown. Arrows indicate centrosomes. Magnification: ×630.
GFP–NPM-NESM was quantified. At least 100 cells were counted for each (g, h) The percentage of mitotic cells that contain detectable NPM signal on
cell-cycle phase. Data are averages of three independent experiments, shown centrosomes (g) or contain more than two centrosomes for each mitotic cell
as mean ± s.d. (c) NPM association with centrosomes in serum-starved (h) was quantified. At least 100 cells were analysed. Data are an average of
HCA2-hTERT cells (G0/G1 phase) was quantified in the presence or absence three separate experiments, shown as mean ± s.d.
containing both murine and human nuclei (Fig. 1b; upper panels). No GFP-positive human nuclei when cells were treated with LMB (Fig. 1b,
human nuclei were positive for GFP–NPM signals when unfused or fused c). These results indicate that NPM can shuttle between the nucleus and
to each other (data not shown). As a control, human nucleolin was not cytoplasm, and the process may depend on a Ran–Crm1 pathway that is
shuttled from human nuclei to murine nuclei for up to 16 h after cell fusion mediated through its NES motif.
(see Supplementary Information, Fig. S1). About 60% of GFP–NPM-posi- To determine whether NPM nucleocytoplasmic shuttling is directed
tive heterokaryons contained GFP-positive human nuclei (Fig. 1c). In by its NES, NLS and NoLS motifs, GFP–NPM mutants were constructed
contrast, less than 10% of GFP–NPM-positive heterokaryons contained by site-directed mutagenesis (Fig. 1d). Wild-type GFP–NPM was
Total protein
(Fig. 1e). These mutant proteins were also found in the nucleolus of cells
pretreated with Triton X-100 to remove soluble NPM signals (data not 100
NPM
shown). These results imply that the NESM and NESD mutant proteins 50
lack the ability to export NPM to the cytoplasm and thus accumulate Crm1 0
in large excess in the nucleus. Mutations at the NLS motif abolished 0 5 10 15 20 25 30
Bottom Top Fraction
localization in the nucleoplasm, as positive staining was mainly found
in the cytoplasm and nucleolus (Fig. 1e). Consistent with a previous LMB (nM)
c
report21, a NoLS mutant at W288G and W290G (NoLSM) was mainly 0 0.2 2 20
found in the nucleus (Fig. 1e), but no longer localized to the nucleolus γ-tubulin
upon Triton pretreatment (data not shown). Consistently, both NESM 100 87 97 74
and NESD mutants lost their ability to shuttle from murine to human NPM
nuclei, whereas both wild type and NoLSM still retained these activities
100 47 23 21
(data not shown). In contrast, NESM-NLS1D was diffusely distributed in
Crm1
both the cytoplasm and the nucleus (Fig. 1e). It appeared that this mutant
100 140 149 79
was not preferentially localized in a specific subcellular compartment
when both import and export motifs were mutated. Taken together, Figure 3 Centrosome-enriched fractions contain NPM, which is sensitive
these results indicate that the NES, NLS and NoLS motifs are functional to the nuclear export inhibitor, LMB. (a) Various fractions obtained from
in directing NPM subcellular localization. discontinuous sucrose gradient centrifugation were analysed by western
blotting with anti-γ-tubulin (top panel), anti-NPM (middle panel) or anti-
Because Crm1 and NPM were recently found to be associated with
Crm1 (bottom panel). Corresponding fraction numbers are indicated.
centrosomes6,15,18, we determined whether their localization required (b) Total protein concentrations of each fraction were determined. (c)
a functional NES motif. Consistent with previous reports15,18, syn- Association of NPM with centrosomes was inhibited by LMB. An aliquot of
chronized G0/G1 cells contained a single centrosome, and over 80% 20 µl of fraction 8 was incubated with various doses of LMB for 30 min,
followed by centrifugation to obtain a centrosome pellet. The pellets were
of these cells also showed a detectable NPM signal on centrosomes
analysed by western blotting with anti-γ-tubulin (top panel), anti-NPM
(Fig. 2a, b). A centrosome-associated NPM signal was absent in inter- (middle panel) and anti-Crm1 (bottom panel). Percentages of the NPM,
phase cells that contained two centrosomes (representing cells in S/G2 γ-tubulin and Crm1 signal intensities, normalized to their untreated
phase) from a non-synchronized population (Fig. 2a, middle panels; controls, are indicated below each blot.
Fig. 2b). In contrast, 100% of mitotic centrosomes showed a detect-
able NPM signal (Fig. 2a, lower panels; Fig. 2b). Similar results were To further examine whether Crm1 and NPM are associated with cen-
obtained in cells expressing GFP–NPM, but not GFP–NPM-NESM trosomes, we enriched centrosomes from quiescent cells by discontinu-
(Fig. 2b). However, greater than 98% of the cells at either interphase ous sucrose gradient fractionation22. Consistent with previous reports15,22,
(containing one or two centrosomes) or mitosis (two centrosomes) fractions 7–9 from HeLa cells, corresponding to 60% (w/w) sucrose, con-
showed detectable GFP–Crm1 on centrosomes (see Supplementary tained the peak signal of γ-tubulin, NPM and Crm1 (Fig. 3a), whereas
Information, Fig. S2A, B). the level of soluble cellular proteins was very low in these fractions
To determine whether regulation of centrosome duplication is (Fig. 3b). On the basis of immunofluorescence staining, only fractions
dependent on NES-mediated NPM transport, serum-starved cells were 7–9 contained centrosomes (see Supplementary Information, Fig. S3B).
either treated with LMB or nucleofected with RanBP1 or HBx. As shown The isolated centrosomes in these fractions retained NPM and Crm1
in Fig. 2c, RanBP1, HBx and LMB completely blocked NPM transport (see Supplementary Information, Fig. S3C); however, no other nuclear
to centrosomes. LMB treatment resulted in a significant decrease in proteins such as nucleolin and a subunit of Ku antigen with relative
the number of cells with a single centrosome (from 90% to 30%), and molecular mass 86,000 (Mr 86K) were detected (see Supplementary
an increase in the number of cells (from 5% to 70%) with more than Information, Fig. S3A). Similar results were obtained from HCA2-
one centrosome (Fig. 2d). Similar results were obtained when another hTERT cells (see Supplementary Information, Fig. S3). Whereas these
bona fide centrosome marker, anti-centrin antibody, was used (see studies do not prove that isolated centrosomes are absolutely free from
Supplementary Information, Fig. S2C, D). In contrast, most of the cells contamination by other cellular proteins, they indicate that fraction 8
treated with LMB still remained in G0/G1, as analysed by fluorescence- is highly centrosome-enriched and that Crm1 and NPM co-separate
activated cell sorting (FACS) (Fig. 2e). Next, we determined whether in this fraction. Following treatment of fraction 8 with LMB, a dose-
LMB acts directly to regulate NPM localization to the centrosome or dependent reduction of the NPM signal was observed (from 100%
indirectly through its effect on nucleocytoplasmic transport. In mitotic to 21%), whereas the levels of Crm1 and γ-tubulin remained similar.
cells, LMB consistently and effectively blocked NPM localization to Taken together, these results suggest that NPM binding to centrosomes
centrosomes, with an increased frequency of mitotic cells containing depends on its NES and is mediated by Crm1. Consistently, an anti-NPM
more than two centrosomes (Fig. 2f–h). Taken together, these results antibody, but not a control mouse IgG, co-immunoprecipitated with
suggest that disruption of Crm1 function by LMB, RanBP1 or HBx leads Crm1 (see Supplementary Information, Fig. S3F).
to NPM dissociation from centrosomes and a subsequent initiation of The results above indicate that LMB-mediated NPM dissociation
premature centrosome duplication. from centrosomes in quiescent cells (G0/G1) may result in unscheduled
a b c
1 1 1
2 2 2
d e f g
B C
NA
40
NA
NA
siR
siR
siR
ol
ol
ntr
ntr
NP
30
Co
Co
NPM 20
100 107 68 18
10
β-actin
0
A2
l
A
tro
RN
iR
on
ls
C
si
tro
PM
on
N
C
Figure 4 Deficiency of NPM by RNA interference is associated with centrosomes. (B) Western blot analysis was used to determine the level of
unscheduled centrosome duplication and abnormal spindles. (A) HCA2- NPM in HCA2-hTERT cells transiently transfected with either a control siRNA
hTERT cells transiently transfected with the NPM siRNA2 oligonucleotides oligonucleotide or two NPM siRNA oligonucleotides for 120 h. The same blot
were co-immunostained either with anti-γ-tubulin (red) and anti-NPM was probed with anti-β-actin antibody as a loading control. Percentages of the
(green) (a–c), or with anti-α-tubulin (red) and anti-γ-tubulin (green) (d–g). NPM signal intensities, normalized to their corresponding β-actin signals and
Nuclei were stained with DAPI (blue) (c, f). Representative interphase cells untreated control, are indicated. (C) The percentage of cells with more than
(a–c) or a mitotic cell (d–g) are shown. Magnification: ×1,000. Panel g is a two centrosomes was determined from HCA2-hTERT cells transfected with
merged image of panels d–f. The inserts 1 and 2 in a are ×5 magnifications or without either control oligonucleotide or NPM siRNA2 oligonucleotides for
of the two areas with centrosomes, as indicated by arrows 1 and 2. One cell 120 h. In the NPM siRNA2 group, only the cells with a diminished expression
(arrow 1) that has a diminished NPM signal contains multiple centrosomes of NPM were evaluated. At least 300 cells were evaluated for each group.
whereas the other cell (arrow 2) with normal NPM expression contains two Data are averages of three independent experiments, shown as mean ± s.d.
centrosome duplication. To further examine the physiological effects of supernumerary centrosomes (more than two centrosomes in each cell)
NPM on centrosome duplication, we used NPM siRNA to knock down were often observed in NPM-siRNA2-transfected cells that were nega-
the expression of endogenous NPM. NPM siRNA2 and to a lesser degree tive for NPM staining (Fig. 4A, a–c; and see Supplementary Information,
NPM siRNA1, but not control siRNA, inhibited NPM expression (Fig. 4A, Fig. S4A). Approximately 30% of NPM-knocked-down cells contained
B). Whereas control cells always contained one or two centrosomes, supernumerary centrosomes (Fig. 4C). Moreover, amplified centrosomes
Cells with n = 1, 2
GFP−NPM which is independent of the essential function of NPM in cell growth.
Endogenous 75
A motif scanning analysis indicates that the threonine residue (T95)
NPM 50
β-actin
within the NES motif of NPM is a potential proline-dependent kinase
25 phosphorylation site. It is possible that phosphorylation at T95 may
FP
Co P−N M
ol S D
NP siR siRN iRNA
NP NPM PM trol
NA
0 alter the NES properties, thereby preventing its binding to Crm1 and
P
siR A + G + G
E
siR
n
+ G P−N
D
NA
NA
FP PM
ol
Co
P
s
ES
docking on centrosomes. To test this hypothesis, we made two addi-
GF
ntr
A
siR
siR
+ G P−N
F
−N
ntr
F
Co
A+
tional mutants of GFP–NPM in which the T95 residue was either
N
ol
M
F
iRN
ntr
NP
siR A + G
NA
N
Co
s
NA
M
N
acid (mimicking phosphorylation at T95). Whereas the GFP-T95A
M
NP
NP siR
M
M
mutant was still able to suppress premature centrosome duplication
M
NP
induced by NPM siRNA, the GFP-T95D mutant partially lost this
ability (Fig. 5c). Consistently, GFP-T95A, but not GFP-T95D, was
efficiently associated with centrosomes (Fig. 5d).
GFP−NPM centrosomes (%)
centrosomes (n>2) (%)
c d
Cells with abnormal
20
100 We recently suggested that the Ran–Crm1 complex may have a role in
15
regulating centrosome duplication because inactivation of Crm1 either
Cells with
75
10 by expression of RanBP1, HBx or by LMB leads to centrosome amplifica-
50
5 tion and multipolar spindles6,8. The effect of HBx on Crm1 is dependent
25
0 on the presence of a NES motif6,13. Consistently, Ran, Crm1 and RanBP1
NA FP FP
NA FP PM
5D
NP siR A + G A + G A
FP 5A
NP NP NP l siRN l
NP siR siR siR A
0
ro
+ G -T9
-T9
siR + G −N
(n = 1) (n = 2)
C
GFP−NPM
or mitotic centriole splitting, an event leading to unscheduled initiation
Co
GFP-T95A
M
M N
cell progression from S to G2 phase, a major portion of NPM is located in Cells and transfection. NIH 3T3, HCA2-hTERT and HeLa cells were main-
the nucleolus, mainly functioning as a regulator of ribosome biogenesis tained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
and cell proliferation23 due to its strong NLS activity. After the nuclear fetal bovine serum, penicillin/streptomycin (1×; 100 U ml−1, 100 µg ml−1), and
200 mM glutamine. GFP–NPM and GFP–NPM mutant plasmids, GFP–Crm1,
membrane breaks down at the beginning of mitosis, NPM reassociates GFP–RanBP1 and pcDNA3-Hbxadr-Hatag13 were transfected into NIH 3T3 or
with mitotic centrosomes through its interaction with the Ran–Crm1 HCA2-hTERT cells using the Nucleofection protocol according to the manu-
complex, preventing centrosome reduplication. Thus, a newly divided G1 facturer’s instructions (Amaxa Biosystems, Gaithersburg, MD). NPM siRNA1
cell contains a NPM-bound centrosome. NPM is then phosphorylated oligonucleotides (target sequence 5′-ccgtcttatttcatttctgta-3′) (Qiagen, Valencia,
and dissociates from the G1 centrosome to initiate centrosome duplica- CA), NPM siRNA2 oligonucleotides (target sequence 5′-atggaatgttatgataggaca-3′)
(Qiagen), and luciferase siRNA oligonucleotides (target sequence 5′-cgtacgcg-
tion and DNA replication. Thus, the Ran network may regulate cell-cycle gaatacttcga-3′) (Dharmacon, Lafayette, CO) were transfected into HCA2-hTERT
progression to ensure faithful cell division24. It is possible that Ran–Crm1 cells with TransIT-TKO transfection reagent (Mirus Corporation, Madison,
may be utilized by other NES-containing proteins such as p53, whose WI) according to the manufacturer’s instructions. The NPM siRNA oligonucle-
stability can be regulated by NPM25,26, or HDM2, a protein containing a otides correspond to the sequences localized in the 3′ UTR of the NPM gene.
functional NES motif20. The oncogenic viral protein HBx binds and inac- Cotransfection of GFP–NPM or GFP–NPM mutants with NPM siRNA was done
using the Nucleofection protocol.
tivates the Ran–Crm1-dependent network6,13, thus disrupting the function
of this complex. This may illustrate a mechanism by which HBx induces Heterokaryon assays. Nucleocytoplasmic shuttling was detected using a heter-
abnormal centrosome duplication and abnormal mitotic spindles, thereby okaryon assay as previously described19,20. Immediately after NIH 3T3 cells in
contributing to HBV-mediated hepatocarcinogenesis27. suspension were nucleofected with GFP–NPM and its mutants, the cells were
seeded on 2-well chambers together with HCA2-hTERT cells. After 20 h, the cells
were treated with 50 µg ml−1 cycloheximide (Sigma, St Louis, MO) in cell culture
METHODS medium for 20 min at 37 °C, and then cell fusion was induced with 50% (w/v)
Plasmid construction. For expression of GFP–NPM, the plasmid pEGFP- polyethylene glycol 8000 (Sigma) in serum-free DMEM for 3 min at 37 °C. The
C2FlagB23 (ref. 28) was kindly provided by K. Nagata (University of Tsukuba, cells were cultured again in cell culture medium containing 50 µg ml−1 cyclohex-
Japan). To generate a plasmid expressing GFP–NPM NESM, pEGFP-C2FlagB23 imide with or without 40 nM of LMB for 2 h, followed by fixing and immunos-
was used as template for two-step overlapping PCR. The following primer pairs taining. Similarly, NIH 3T3 cells were nucleofected with GFP–PML, fused to
were used for the first rounds of PCR: (a) 5′-acacttcgcccttgcgaccactggtggtg-3′ HCA2-hTERT cells, and cultured for up to 16 h in culture medium containing
and (b) 5′-tcgaattctgcagtcgac-3′; and (c) 5′-gtcgcaagggcgaagtgtggttcagggccag- 50 µg ml−1 of cycloheximide, followed by fixing and immunostaining.
3′ and (d) 5′-tgatcagttatctagatcc-3′. The products of these reactions were then
purified, annealed and used as a template for a final PCR using primers (b) Cell-cycle analysis. Serum-starved HCA2-hTERT cells used in NPM centro-
and (d). The final PCR product was digested with BamHI and subcloned into somal localization assays were also evaluated by cell-cycle analysis. Cells were
the pEGFP-C2FlagB23 cut with BamHI. Similarly, GFP-T95A was generated trypsinized, washed twice with phosphate-buffered saline, pelleted, and fixed
using two-step overlapping PCR with primer pairs (b) and 5′-taagaccactggt- with 70% ethanol overnight at 4 °C. Samples were pelleted, washed once with
ggtgctatttcaaag-3′, and (d) and 5′-tagcaccaccagtggtcttaag-3′ for first rounds PBS, treated with 10 µg ml−1 RNaseA and stained with 50 µg ml−1 of propidium
of PCR, and primers (b) and (d) for final PCR. The final PCR product was iodide for 30 min at 37 °C, and then subjected to FACScan analysis.
digested with BamHI and subcloned into pEGFP-C2FlagB23 at the BamHI site.
GFP–NPMT95D was generated with primer pairs (b) and 5′-taagaccactggtgggtc- Cell proliferation assay. GFP–NPM plasmid and NPM siRNA co-transfected
tatttcaaag-3′, and (d) and 5′-tagacccaccagtggtcttaag-3′ for first rounds of PCR, HCA2-hTERT cells were frozen at −70 °C at different time points. The frozen cells
and primers (b) and (d) for final PCR. The final PCR product was digested with were thawed and subjected to a cell proliferation assay using the CyQUANT assay
BamHI and subcloned into the pEGFP-C2FlagB23 cut with BamHI. To gener- according to the manufacturer’s instructions (Molecular Probes, Eugene, OR).
ate the GFP–NPM NESD expression plasmid, PCR fragments from pEGFP-
C2FlagB23 with primers (b) and 5′-tggtgttaagcttcaaagcccccaag-3′ cut with Centrosome isolation. Centrosomes were prepared from quiescent HeLa and
KpnI and HindIII, and PCR fragments from pEGFP-C2FlagB23 with primers HCA2-hTERT cells as described22. In brief, HeLa or HCA2-hTERT cells were
(d) and 5′-ttaaggtaagcttgtggttcagggccag-3′ cut with BamHI and HindIII were cultured for 2 days, followed by serum starvation for 24 h. Cells were pelleted,
ligated into the pEGFP-C2FlagB23 digested with BamHI and KpnI. To gener- washed with TBS and TBS 1/10–8% sucrose buffer, lysed and subjected to cen-
ate the GFP–NPM NLS1D expression plasmid, PCR fragments from pEGFP- trifugation at 2,500g for 10 min to remove cell debris, nuclei and chromatin. The
C2FlagB23 with primers (b) and 5′-ggcagaaagcttcacatcctcctcctcttc-3′ cut with supernatant was filtered with a nylon mesh and treated with 2 U ml−1 DNase I,
PstI and HindIII, and PCR fragments from pEGFP-C2FlagB23 with primers followed by centrifugation with a 60% sucrose cushion at the bottom of centrifu-
(d) and 5′-gatgtgaagctttctgcccctggaggtggt-3′ digested with BamHI and HindIII gation tube for 30 min at 11,000g. The centrosomes in the sucrose cushion were
were ligated into the pEGFP-C2FlagB23 cut with BamHI and PstI. To gener- diluted with lysis buffer and subjected to a discontinuous gradient centrifugation
ate the GFP–NPM NLS2D expression plasmid, PCR fragments from pEGFP- consisting of 2.5 ml of 70%, 1.5 ml of 50% and 1.5 ml of 40% sucrose solution at
C2FlagB23 with primers (b) and 5′-agcagcaagctttacgctaccacctccaggggcagc-3′ 85,000g for 1.5 h with a SW40Ti rotor. Centrosome fractions (300 µl per fraction)
cut with PstI and HindIII, and PCR fragments from pEGFP-C2FlagB23 with were collected from the bottom of a centrifugation tube. Protein concentrations
primers (d) and 5′-aaagtaaagcttgctgctgatg-3′ cut with BamHI and HindIII of each fraction were determined by the Bio-Rad protein assay dye reagent (Bio-
were ligated into the pEGFP-C2FlagB23 cut with BamHI and PstI. To gener- Rad, Hercules, CA) according to the manufacturer’s instructions. The fractions
ate the GFP–NPM NoLSM expression plasmid, PCR fragments from pEGFP- containing centrosomes were determined by immunoblot analysis for γ-tubulin,
C2FlagB23 with primers (b) and 5′-tccggtggatccttaaagagacttcctcccctgcccgag-3′ and immunofluoescence for γ-tubulin and α-tubulin as described22. The aliquots
cut with BamHI were subcloned into the pEGFP-C2FlagB23 cut with BamHI. from the centrosome fractions were diluted with 1 ml of 10 mM PIPES buffer
To generate the GFP–NPM NESM-NLS1D expression plasmid, PCR fragments (pH 7.2) and subjected to LMB (0.2, 2 or 20 nM) treatment for 30 min at room
from pGFP-NPM NLS1D plasmid with primer pairs (a) and (b), and (c) and (d) temperature, followed by centrifugation at 20,000g for 10 min. Pellets were washed
were purified, annealed and used as a template for a final PCR using primers three times in 10 mM PIPES. Proteins associated with LMB-treated centrosomes
(b) and (d). The purified product was cut with BamHI and subcloned into the were then detected by immunoblot analysis.
pEGFP-C2FlagB23 digested with BamHI. To generate the GFP–NPM NLS1/2D
expression plasmid, a fragment of GFP–NPM NLS1D digested by KpnI and Immunoblot analysis. HCA2-hTERT cells were homogenized in NP40 lysis
HindIII and a fragment of GFP–NPM NLS2D cut by BamHI and HindIII were buffer (1% NP40, 25 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM
ligated in the pEGFP-C2FlagB23 plasmid digested with BamHI and KpnI. sodium orthoVanadate, 100 µM GTPγS and protease inhibitors), and subjected to