Thesis: Debanjan Goswami

Modes of nano-scale clustering of GPI-anchored
protein at the cell surface.
A Thesis
Submitted to the
Tata Institute of Fundamental Research
Mumbai, India
Thesis © 2009 Debanjan Goswami
for the degree of

Doctor of Philosophy
in
Cell Biology
By
Debanjan Goswami
National Centre for Biological Sciences

Tata Institute of Fundamental Research
Bangalore, India.
2009
ii
© 2009 Debanjan Goswami
Declaration
This thesis is a presentation of my original research work.

Wherever contributions of others are involved, every effort is made to
indicate this clearly, with due reference to the literature, and
acknowledgement of collaborative research and discussions.
The work was done under the guidance of Professor Satyajit

Mayor, at the National Centre for Biological Sciences - Tata Institute of
Fundamental Research, Bangalore, India.
Debanjan Goswami,
Candidate, NCBS-TIFR
Bangalore, India, 560065.
In my capacity as supervisor of the candidate’s thesis, I certify

that the above statements are true to the best of my knowledge.
Prof. Satyajit Mayor

Supervisor, NCBS-TIFR,
Bangalore, India, 560065.
iii
… dedicated to my parents,
who taught me the best kind of knowledge
and upheld my stamina to keep up my best.
iv
Acknowledgements
I have tried to consolidate in this section the reminiscence from my

journey to this stage of my career. Here, I deeply acknowledge and convey my
sincere thanks to those people who were both for being significant and important
to my life.
During my graduate studies, my thesis supervisor, Jitu, contributed

substantially, by giving me the opportunity to pursue research in his laboratory.
This generosity and opportunity to me was seasoned with thorough and rigorous
guidance. He has always given me the liberty to do experiments of my choice and

at the same time, supported me to streamline those as additions to the story.
Inspiration always came from his one liner – ‘the learning curve should always be
exponential and never saturate’. He is very enthusiastic and energetic and he
always managed to put enormous effort for any scientific discussion. Apart from
our day-to-day project discussions, I have encountered many instances where he
made me to grow up as a person – more specifically as a logical thinker.
My thesis committee members Dr. Madan Rao, Dr. Mathew K. Mathew

and Dr. Sudipta Maiti were always very active, critical and enthusiastic about my
progress in research. Specially, while setting up microscopes, Sudipta-da was
involved right from the beginning and guided me step-by-step. His knowledge in
instrumentation and perception in troubleshooting was always helped me figuring
out problems with the same. Nonetheless, he gave me insights about data analysis
and never stood back from basic criticism of my research. Madan caught hold of
me when I got puzzled with enigmatic biophysical behavior of the cellular plasma
membrane. He always had for biological problems smart applying the explanation
from basic physics. He is fantastic person. He is another reason that I could
perceive draft explanations for unresolved biological phenomena seen in my
experimental results. In many instances he also swerved many of my future
v
Acknowledgements
experiments in the right direction. Mathew (Prof. Mathew K. Mathew) was
always very generous and patient with my projects. He added many important
suggestions regarding fluorescence phenomena in biological samples. I want to
thank Prof. G. Krishnamoorthy from DCS, TIFR for introducing me to time-
resolved fluorescence dynamics in his lab that obviously was a great consequence
for my research later on. Prof. N. Periasamy DCS, TIFR helped me to
understand the fitting routine for time-resolved fluorescence dynamics and has
always been supporting me for any modifications required in analysis program. I
thank Dr. Kulkarni, JNCASR, India for allowing me to work in his laboratory to
make nano-gold particles. I would like to acknowledge Prof. Jim Spudich,
Stanford Univerisity, USA for his criticism, useful suggestions and discussions
during our collaboration. I am grateful to Dr. H. Krishnamurthy (CIFF, NCBS)
for his constant support and useful suggestions while dealing with the problems
in multiple microscopes; actually, I enjoyed whole 5-years working right beside
Krishna. I acknowledge Dr. Gaiti Hasan and Dr. Apurva Sarin as head of
academics. I would like to thank Prof. Vijayraghavan, the director of NCBS, for
helping me to frame and find future scientific research opportunities.
Kripa G., Madan’s student, deployed all necessary tools to couple the
theoretical explanations with our experimental data. She is brilliant. Since I met
her, she has always been more than just a collaborator but a good friend for
hanging out and discussing science in general.
Sameera Bilgrami, the person from whom I learned cell culturing,

microscope handling and wide field anisotropy measurements. I would like to pay
my sincere regards to her for being thorough and rigorous; also for being there
during the initial course of my novice footsteps in to the laboratory. Sriram who
inspired, criticized and nurtured me to grow up in many aspects especially related
to science. I convey my respect to him. My first collaboration with David Altman
at second year was an extraordinary experience for me. I learnt a lot from him
while doing experiments and data analysis. Along with unlimited fun with Gagan,
he encouraged me and helped me to improve scientific writing and computer
vi
Acknowledgements
skills. Without Rahul, Manjula and Abhijit (the ‘Kale from Pune’) I can’t even
think of the fulfillment of entertainment in the lab. Me and Neha started working
together for a collaborative project. Precisely, it was an amazing experience for
me. I thank Riya for her effort to make necessary probe that made my
experiments feasible and Sanat from RRI for teaching me lipid related work. I
acknowledge my batch mates for making me participate in the coursework
discussions and all the events and especially Sudha, for all the interesting
interactions in the lab. I enjoyed working with Swetha and Subhasri in the lab. In
the recent past, Suvrajit started working with me and it is a different experience
while passing on some of the expertise. I liked working with him for his aptitude
and analytical understanding. I thank all the present members in the lab for
keeping the lab environment rather cool and cozy. It is difficult to write about
Mr. Gautam Dey – the cool and bright dude in the lab. I lived it up many
evenings with him and certainly, will do in future. I acknowledge Shanta and
Vishalakshi for prompt help with the official and/or academic matter; Mr. Ashok
Rao for all accounts related issues; Ranjith, kitchen stuff and Nagaraj for keeping
up an efficient system to bring us research facilities and materials promptly. I
cannot forget about my seniors, Santanu-da, Samarjit-da and Saikat-da for their
continuous support at multiple levels and especially, because with them NCBS
was a home away from home. I admire them for all the useful and critical
suggestions. I would like to thank Deepak, Bidisha, Feroz and Aprotim from
Shiva’s Lab for teaching me scientific programming in LabView, discussion on
different type of experimental and instrumental problems. I saved the most
important for the last; I am very lucky to have a friend with me and cooled whale
as part of our graduate life.
I found the desired approach towards the interdisciplinary field of science

during my master’s program in Biophysics at Rajabazar Science College, Calcutta
University. I want to express my gratitude to faculties Prof. Chanchal K.
Dasgupta, Prof. Uma Dasgupta, Prof. Utpal Chaudhuri, Prof. Ashok R. Thakur,
Dr. Subhasish Mukherjee for aiding my interest into a more research oriented
arena of scientific studies. And it goes without saying that the world here in
vii
Acknowledgements
NCBS been impossible for me without their earnest effort through the rigorous
and rich content based interdisciplinary course that they so willfully provided us.
Definitely, the whole MSc program wouldn’t have been so much fun without all
my classmates; spent good times during scientific discussions, projects and
ofcourse the annual departmental picnic. During my MSc course, I got the
opportunity to do a summer research project at IMTECH, Chandigarh under Dr.
Purnananda Guptasharma. I am very glad to have had Sourav Mukherjee in that
laboratory with whom I experienced the essence of scientific research for the first
time.
Entering undergrad college and choosing Microbiology honours course

was a big decision for me. It was a fairly new interdisciplinary course in Calcutta
University. It did modify the orientation and foundation of my understanding of

biological sciences. I would like to acknowledge faculty members of the college
including Prof. G. Bhattacharya, Dr. Swapna Mukhrejee, Dr. Karuna Baneerjee
Dr. Sathi Das and Dr. Tania Das. Debashis Mukherjee, the guiding star from
Saha Institute of Nuclear Physics, Kolkata who enlighten the path to explore
microbiology as more than just a textbook science.
In my high school, Jadavpur Vidyapith, a first grade Bengali medium

school in south Kolkata, I came across a bunch of marvelous teachers including
my father who helped me build a strong fundamental understanding of different
subjects. I would like to express my tribute to all of them. Remembering school
days, such a fabulous time I spent with all my friends; specially, Subhasish
Chakraborty, with whom life did not end at crazy times and fun stuff, we also
always strangely managed to complement each other academically. With
Devdeep, in high school and in college days, it was different. As he was obsessed
with physics and I being passionate, we enjoyed studying physics together. He
helped me to understand and appreciate the subject, the way it meant to be.
After I got to know Debalina, my wife (not then… duh!! I don’t support
child marriage), I felt myself to be the luckiest. From college through university,
viii
Acknowledgements
she was always with me and inspired me in my good and bad times. I was always
very passionate discussing science, history and literature with her and it still
intrigues me that the desire to do so hasn’t come across an inch closer to being
extinguished. She is simply fantastic and the only person who comforts me in all
aspect of life. I am grateful to her for being in my life and she will always be an
inspiration to me.
My parents, the reason for my existence and whose contribution cannot

be put into words, have always given unconditional love and support without
which I would not believe in myself enough to be who I am. Specially, they
taught me the sense of perfection, honesty, hard work, ethics in life and the
aptitude for knowledge. They always allowed me to choose my career. They
trusted my independent decisions and they have confidence in me. My sister, a

lively personality, has always been able to bring me smile anytime. My
grandparents, with whom I partly grew up in my childhood, are esteemed
monuments of inspiration in my life. They are very proud of me not only because
of my achievement but my constant effort to be better myself. I want to convey
my reverence to them. My eldest uncle, who expanded my horizon in terms of
general knowledge and education at my young age, would be the happiest person
seeing me graduate with PhD degree. I would take this opportunity to thank
other family members, all my uncles, aunts and cousins for bringing me love and
fun that could not have been brought to me by any other means. I feel privileged
for having such a nice family.
ix
Table of Contents
DECLARATION ...................................................................................................III
ACKNOWLEDGEMENTS....................................................................................V
TABLE OF CONTENTS ......................................................................................X
CHAPTER 1 ............................................................................................................... 1
INTRODUCTION ....................................................................................................... 1
1.A: SUMMARY ....................................................................................................... 1
1.B: BACKGROUND ................................................................................................... 2
1.B.i: Structure of plasma membrane ............................................................... 2

1.B.ii: The GPI-anchored proteins and its discovery ........................................... 3
1.B.iii: The structure and biosynthesis of GPI-APs.............................................. 4
1.B.iv: The membrane association of GPI-APs ................................................... 5
1.C: STATE OF THE MEMBRANE – ‘RAFT HYPOTHESIS’ ...................................................... 5
1.C.i: Artificial membrane – description of ‘rafts’ .............................................. 5
1.C.ii: Rafts in biological membrane redefined .................................................. 7
1.D: BIOPHYSICAL METHODS....................................................................................... 8
1.E: OBJECTIVE OF THE THESIS................................................................................... 12
1.F: REFERENCES.................................................................................................... 13
FIGURE 1.A GPI-ANCHOR AND ANCHORED PROTEINS ..................................................... 16
FIGURE 1.B CONCENTRATION INDEPENDENT ANISOTROPY AT CELL SURFACE ........................ 17
FIGURE 1.C PICTORIAL REPRESENTATION OF GPI-ANCHORED PROTEIN ORGANIZATION .......... 18
CHAPTER 2 ............................................................................................................. 19
MATERIALS AND METHODS ................................................................................... 19

2.A: Cell lines .................................................................................................. 19
2.B: Cell culture media and imaging buffer ..................................................... 20
2.C: FLUORESCENCE LABELING ON CELL SURFACE ........................................................... 20
2.D: Cholesterol depletion procedures ............................................................ 21
2.E: Actin or myosin perturbing procedures .................................................... 21
2.F: Wide field microscopy .............................................................................. 22
2.G: Confocal microscopy ............................................................................... 24
2.H: REFRERENCES ................................................................................................. 25
FIGURE 2.A SATURATION BINDING ASSAY FOR PLF PROBE ............................................... 26
FIGURE 2.B IMAGES OF GG8 CELLS ............................................................................ 27
FIGURE 2.C WIDE-FIELD ILLUMINATION PROFILE............................................................ 28
FIGURE 2.D MEASURING SMALL SYSTEMATIC CHANGES OF ANISOTROPY VALUE .................... 29
x
Contents
CHAPTER 3 ............................................................................................................. 30
MICROSCOPES, INSTRUMENTATION AND ANALYTICAL METHODS FOR FRET-BASED
MEASUREMENTS ON CELLS.................................................................................... 30
3.A: INTRODUCTION ............................................................................................... 30

3.A.i: Anisotropy-based FRET measurements .................................................. 30
3.A.ii: Fluorescence lifetime based FRET measurements.................................. 32
3.B: MULTIPHOTON LASER SCANNING CONFOCAL MICROSCOPE ........................................ 32
3.B.i: Microscope setup ................................................................................. 32
3.B.ii: Advantages of multiphoton excitation .................................................. 34
3.B.iii: Time-resolved fluorescence measurements from living cells using TCSPC
830 card. ........................................................................................................ 35
3.B.iv: Time-resolved fluorescence studies on live cells .................................... 39
3.B.v: Time-resolved fluorescence decay analysis............................................ 41
3.B.vi: Steady-state anisotropy measurements using TCSPC 830 card. ............ 45
3.B.vii: Steady-state anisotropy analysis ........................................................ 45
3.C: LINE-SCANNING CONFOCAL MICROSCOPE .............................................................. 46

3.C.i: Microscope setup .................................................................................. 46
3.D: HETERO-FRET MEASUREMENTS ......................................................................... 48
3.D.i: Theory................................................................................................... 48
3.D.ii: Calculation of R 0 .................................................................................. 50
3.D.iii: Measurement of FRET efficiency .......................................................... 51
3.D.iv: Hetero FRET measurements by donor lifetime...................................... 52
3.D.v: Varying donor to acceptor ratio – Hetero FRET efficiency measurement 54
3.E: REFERENCE ..................................................................................................... 55
FIGURE 3.A MICROSCOPE SET UP FOR FIAT AND TIME-RESOLVED FLUORESCENCE
MEASUREMENTS..................................................................................................... 57
FIGURE 3.B ILLUMINATION PROFILE FOR SINGLE PHOTON AND MULTIPHOTON FLUORESCENCE
PROCESS ............................................................................................................... 58
FIGURE 3.C CONFIRMATION OF TWO PHOTON EXCITATION .............................................. 59
FIGURE 3.D INITIAL ANISOTROPY VALUES FOR DIFFERENT MODE OF EXCITATION ................... 60
FIGURE 3.E PRINCIPLE OF TIME CORRELATED SINGLE PHOTON COUNTING (TCSPC) ............... 61
FIGURE 3.F INSTRUMENT RESPONSE FUNCTION (IRF) ..................................................... 62
FIGURE 3.G MULTIPHOTON ILLUMINATION PROFILE ....................................................... 63
FIGURE 3.H EXAMPLES OF TIME- RESOLVED FLUORESCENCE DECAYS .................................. 64
FIGURE 3.I LINE-SCANNING CONFOCAL LSM 5 LIVE MICROSCOPE...................................... 65
CHAPTER 4 ............................................................................................................. 66
AEROLYSIN TOXIN ALTERS GPI-ANCHORED PROTEIN ORGANIZATION AT THE CELL
SURFACE ................................................................................................................ 66
4.A: INTRODUCTION ............................................................................................... 66
4.B: RESULTS ........................................................................................................ 69
4.B.i: Uniform Surface Distribution of GPI-APs ................................................ 69
4.B.ii: Aerolysin induces alteration in GPI-AP organization on cell surface ....... 70
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Contents
4.B.iii: Aerolysin induced GPI-AP clusters are compact .................................... 71
4.B.iv: Confirmation of higher order organization with fluorescence lifetime
measurements ............................................................................................... 71
4.B.v: Estimation of cluster size and fraction can be done by theoretical
modeling of hetero-FRET ................................................................................ 72
4.C: DISCUSSION .................................................................................................... 76
4.D: REFERENCES ................................................................................................... 77
TABLE 4.A: TRA DATA FOR HOMO-FRET MEASUREMENT................................................ 80
TABLE 4.B: DONOR FLUORESCENCE LIFETIME WITH VARYING A/D ..................................... 81
FIGURE 4.A: PROBABLE STRUCTURE OF AEROLYSIN COMPLEX ........................................... 82
FIGURE 4.B: DISTRIBUTION OF GPI-ANCHORED PROTEIN ON CELL SURFACE ......................... 83
FIGURE 4.D: TIME RESOLVED ANISOTROPY DECAYS FOR GPI-AP ORGANIZATION .................. 85
FIGURE 4.D: TIME RESOLVED ANISOTROPY DECAYS FOR GPI-AP ORGANIZATION .................. 85
FIGURE 4.E: HETERO FRET OBSERVED BY FLUORESCENCE LIFETIME ................................... 86
FIGURE 4.F: ENERGY TRANSFER EFFICIENCY CHANGES WITH D TO A RATIO .......................... 87
FIGURE 4.F: ENERGY TRANSFER EFFICIENCY CHANGES WITH D TO A RATIO .......................... 87
CHAPTER 5 ............................................................................................................. 88
GPI-ANCHORED PROTEIN NANO-CLUSTERS ARE IMMOBILE AND
HETEROGENEOUSLY ON LIVING CELL MEMBRANE................................................. 88
5.A: INTRODUCTION ............................................................................................... 88
5.B: RESULTS ........................................................................................................ 91
5.B.i: GPI-AP clusters are preferentially distributed in certain regions of the cell
surface ........................................................................................................... 91
5.B.ii: Non-random distribution of sub-resolution clusters of GPI-AP on flat
cellscapes....................................................................................................... 92
5.B.iii: Nanosclusters are immobile ................................................................. 94
5.B.iv: Actin perturbation affects nano-cluster reformation – further confirms
nanoclusters are immobile ............................................................................. 95
5.B.v: Formation of nanocluster is sensitive to levels of cholesterol at the
plasma membrane ......................................................................................... 96
5.C: DISCUSSION .................................................................................................... 97
5.D: References .............................................................................................. 99
VARMA, R., AND S. MAYOR. 1998. GPI-ANCHORED PROTEINS ARE ORGANIZED IN SUBMICRON
DOMAINS AT THE CELL SURFACE. NATURE. 394:798-801.FIGURE 5.A SPATIAL DISTRIBUTION OF
NANOCLUSTERS .................................................................................................... 100
FIGURE 5.A SPATIAL DISTRIBUTION OF NANOCLUSTERS ................................................. 101
FIGURE 5.B STATISTICAL ANALYSIS OF DISTRIBUTION OF NANOCLUSTER ............................ 102
FIGURE 5.C ANISOTROPY RECOVERY AFTER PHOTOBLEACHING AT 20°C IMAGES................ 103
FIGURE 5.D GRAPH SHOWS QUANTIFICATION OF ARAP DATA AT 20°C ............................ 104
FIGURE 5.E ANISOTROPY RECOVERY AFTER PHOTOBLEACHING AT 37°C IMAGES................ 105
FIGURE 5.F GRAPH SHOWS QUANTIFICATION OF ARAP DATA AT 37°C ............................ 106
FIGURE 5.G GRAPH SHOWS QUANTIFICATION OF ARAP DATA AFTER LATRUNCULIN AND
BLEBBISTATIN TREATMENT ...................................................................................... 107
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Contents
FIGURE 5.H GRAPH SHOWS QUANTIFICATION OF ARAP DATA AFTER METHYL Β-CYCLODEXTRIN
........................................................................................................................ 108
CHAPTER 6 ........................................................................................................... 109
CORTICAL ACTIN DRIVEN STEADY STATE DYNAMICS OF GPI-AP MONOMERS AND

NANOCLUSTERS – AN ACTIVE PROCESS ............................................................... 109
6.A: INTRODUCTION ............................................................................................. 109
6.B: RESULTS ...................................................................................................... 110
6.B.i:Assay to study steady-state dynamics of GPI-AP organization at the cell
surface ......................................................................................................... 110
6.B.ii: Lipid shows typical concentration dependent FRET signal.................... 110
6.B.iii: GPI-AP nanoclusters remain immobile at the scale of confocal area and
follows unusual interconversion. .................................................................. 111
6.B.iv: Temperature dependence of the dynamics......................................... 112
6.B.v: Spatial heterogeneous nature of association-dissociation kinetics at the
cell surface irrespective of temperature. ....................................................... 113

6.B.vi: Non-Arrhenius interconversion dynamic............................................. 113
6.B.vii: Cholesterol-sensitive interconversion ................................................ 114
6.B.viii: Role of cortical actin in interconversion ............................................ 114
6.C DISCUSSION ................................................................................................... 117
6.D: REFERENCES ................................................................................................. 118
TABLE I: HETEROFRET+ MEASUREMENT BY DONOR FLUORESCENCE LIFETME..................... 119
TABLE II: TIME RESOLVED HOMOFRET MEASUREMENTS ............................................... 121
FIGURE 6.A IMAGING AND INTERCONVERSION DYNAMICS ASSAY ..................................... 123
FIGURE 6.B FIAT ASSAY FOR BODIPY-SM AT CELL SURFACE ......................................... 124
FIGURE 6.C INTENSITY AND ANISOTROPY TRACES AND IMAGES FROM CELL SURFACE-LABELED
GPI-APS AT 20°C. ............................................................................................... 125
FIGURE 6.D INTENSITY AND ANISOTROPY TRACES AND IMAGES FROM CELL SURFACE-LABELED
GPI-APS AT 37°C. ............................................................................................... 126
FIGURE 6.E SCHEMATIC REPRESENTATION OF FIAT ASSAY ON CELL SURFACE...................... 127
FIGURE 6.F SCHEMATIC OF DYNAMICS OF GPI-ANCHORED PROTEINS ............................... 128
FIGURE 6.G EXAMPLES OF THEORETICAL FITS OBTAINED FROM FIAT ASSAY ....................... 129
FIGURE 6.H RATE OF DIFFUSION OF MONOMERS OBTAINED FROM THE FIT ........................ 130
FIGURE 6.I INTERCONVERSION RATES OBTAINED FROM THE FIT ....................................... 131
FIGURE 6.J TEMPERATURE DEPENDENCE OF INTERCONVERSION RATES ............................. 132
FIGURE 6.K PERTURBATION OF CHOLESTEROL LEVELS IN MEMBRANE ALTERS DYNAMICS....... 133
FIGURE 6.L BLEBS ARE DEVOID OF ACTIN.................................................................... 134
FIGURE 6.M HETERO-FRET MEASUREMENTS BY DONOR FLUORESCENCE LIFETIME: BLEBS ARE
DEVOID OF NANOCLUSTERS ..................................................................................... 135
FIGURE 6.N FLIM DATA SHOWS BLEBS HAS LESS HETERO-FRET COMPARED TO FLAT PART OF CELL
........................................................................................................................ 136
FIGURE 6.O TIME-RESOLVED ANISOTROPY DATA FOR HOMO-FRET MEASUREMENT ............ 137
FIGURE 6.P NANOCLUSTERS ARE NOT PRESENT IN MEMBRANE STRUCTURE DEVOID OF ACTIN
(BLEBS) AS SEEN IN FIAT EXPERIMENTS...................................................................... 138
FIGURE 6.Q ACTIN PERTURBATION INFLUENCES NANOCLUSTER FORMATION ...................... 139
FIGURE 6.R INHIBITION OF MYOSIN ACTIVITY INFLUENCES NANOCLUSTER FORMATION ......... 140
xiii
Contents
CHAPTER 7 ........................................................................................................... 141

CONCLUSIONS AND DISCUSSION ......................................................................... 141
Conclusions: ................................................................................................. 141
Discussion: ................................................................................................... 142
REFERENCES: ....................................................................................................... 143
PUBLICATIONS ..................................................................................................... 145
SYNOPSIS ............................................................................................................. 146

xiv
Chapter 1
Introduction
1.A: Summary
A hallmark in the understanding of cell membrane organization

and structure was encapsulated in the Fluid Mosaic model (Singer and
Nicolson, 1972), where the membrane was visualized as an
equilibrated two-dimensional fluid – a passive mixture of proteins
dissolved in a sea of lipids. According to this model, all lipids and
proteins (ratio varies from 1:4 to 4:1) diffuse freely at all length-scale on
the surface of the cell (Frye and Edidin, 1970). Over the last decade,
the concept of a compartmentalized membrane has emerged where
the cell surface is not a homogeneous mixture, but is segregated into
domains. The mechanism of formation and maintenance of such
domains is hypothesized as arising due to the interaction between
specific lipids such as cholesterol and sphingolipids and associated
proteins. Compartmentalized regions or domains on cell membrane are
referred as ‘lipid-rafts’. ‘Lipid-rafts’ are proposed to be involved in a
variety of important biological roles including endocytosis, trafficking,
signaling complex formation (Simons and Ikonen, 1997). Although
numerous biological functions have been ascribed to ‘lipid-rafts’, the
mechanism behind their formation, structure and dynamics remain
highly debated (Mayor and Rao, 2004).
1
Introduction
1.B: Background
1.B.i: Structure of plasma membrane
The plasma membrane is assembled from a variety of lipids

(which may be broadly classified as phosphoglycerides, sphingolipids,
and sterols) and proteins. All three classes of lipids are amphipathic
molecules having a polar (hydrophilic) head group and hydrophobic
tail. The hydrophobic effect and van der Waals interactions cause the
tail groups to self-associate into a micelle or a liposome or a bilayer
with the polar head groups oriented toward water. Although the
common membrane lipids have this amphipathic character in common,

they differ in their chemical structures, abundance, and functions in the
membrane. Phosphoglycerides, the most abundant class of lipids in
most membranes, are derivatives of glycerol-3-phosphate. A typical
phosphoglyceride molecule consists of a hydrophobic tail composed of
two fatty acyl chains esterified to the two hydroxyl groups in glycerol
phosphate and a polar head group attached to the phosphate group.
The two fatty acyl chains may differ in the number of carbons that they
contain (commonly 16 or 18) and their degree of saturation (0, 1, or 2
double bonds). The second class of membrane lipids, sphingolipids, is
derived from sphingosine, an amino alcohol with a long hydrocarbon
chain, and contains a long-chain fatty acid attached to the sphingosine
amino group. Sterols, the third important class of membrane lipids,
consist of cholesterol and its derivatives. The basic structure of
cholesterol is a planar four-ring hydrocarbon with polar hydroxyl group.
The proteins in the plasma membrane are either partially or completely
integrated (known as integral membrane proteins) or loosely attached
with the polar head group of specific lipids (peripheral membrane
proteins).
2
Introduction
The lipid and protein profile of membranes varies across cell
type, age, health and also depends on the two leaflets of a particular
bilayer. Differences in lipid composition may also correspond to the
specialization of membrane function. For example, the plasma
membrane of absorptive epithelial cells lining the intestine exhibits two
distinct regions: the apical surface faces the lumen of the gut and is
exposed to widely varying external conditions; the basolateral surface
interacts with other epithelial cells and with underlying extracellular
structures. However, how lipid compositions are modulated and
maintained by the cell and that lead to different physiological processes
is yet to resolve. The role of lipid composition and heterogeneity play in
various endocytic and signaling functions in a cell is unknown.
1.B.ii: The GPI-anchored proteins and its discovery
Amongst several peripheral membrane proteins,

Glycosylphosphatidyl Inositol Anchored Proteins (GPI-APs) is one of
the interesting lipid anchored protein on the cell membrane. Various
enzymes, receptors, signaling molecules, adhesion molecules, cell
surface antigens and prion proteins fall into this category (Paulick and
Bertozzi, 2008). The GPI anchored proteins are attached to only the
outer leaflet of the bilayer unlike other integral membrane proteins via
phospholipids, a part of the GPI moiety. A novel phospholipase that
cleaves phosphatidylinositol was purified in 1976 from Bacillus cereus.
This phospholipase was named phosphatidylinositol phospholipase C
(PIPLC). Subsequently it was shown that purified phopholipase C can
completely remove all the alkaline phophatase activity from membrane
pellets specifically (Low and Finean, 1977) . By 1985, the structural
components of the GPI-APs were identified by multiple
chromatography techqniques from couple of protozoan (Trypanosoma
3
Introduction
brucei) surface proteins, variant surface glycoprotein (VSG), and a
glycoprotein expressed on mammalian thymocytes, Thy-1.
1.B.iii: The structure and biosynthesis of GPI-APs
Following their discovery, extensive research commenced to

elucidate the structure of the GPI anchor, biosynthesis of the GPI-APs,
and their localization in cells and has been comprehensively described
in a number of reviews (Chatterjee et al., 2001). Briefly, the signal
peptide is recognized, cleaved and replaced by pre-assembled GPI by
the action of a GPI transamidase residing in the endoplasmic reticulum
(ER). The GPI transamidase is a membrane-bound multi subunit

enzyme containing several PIG gene products which add each
component of the GPI anchor. As we can describe the GPI-anchor, the
membrane anchoring is done by two fatty acyl chains linked to a
glycerol backbone. The third hydroxyl of the glycerol esterified to a
phospho-inositol group that is further linked to an oligosaccharide
consisting of glucosamine and mannose residues and a terminal
phosphoethanolamine that is linked to the carboxy-terminal cysteine of
the protein (Figure 1.A).
The GPI anchor is synthesized with an unsaturated fatty acyl

chain at sn2 position and has a palmitate linked to inositol. Deacylation
of inositol is required for further maturation of the anchor in the Golgi
and is achieved by the deacylase, PGAP1. PGAP3 deacylates the
unsaturated fatty acyl chain at sn2 and PGAP2 reacylates it with
stearic acid (Tanaka et al., 2004). The reason behind the inefficient
process cells adapted for additional deacylation and reacylation
reactions is to ensure that both the fatty acyl chains attached to the
glycerol are saturated.
4
Introduction
The lipid structure is important for its transport and surface
association. In PGAP1-defective CHO cells, when inositolacylated GPI-
APs were expressed (Tanaka et al., 2004), transport of inositol-
acylated GPI-APs from the ER to the Golgi apparatus was at 4-fold
reduced rate compared to that of normal GPI-APs in the wild-type CHO
cells.
1.B.iv: The membrane association of GPI-APs
A well popular notion about GPI-APs is its putative association

with phase segregated membrane domains enriched in selective lipids,
such as sphingolipids and cholesterol, termed ‘rafts’. As shown

previously, GPI-APs are associated with detergent-resistant membrane
(DRM) fraction when cells are extracted with cold non-ionic detergent,
such as Triton X-100 (Brown and Rose, 1992; Mayor and Riezman,
2004). Glycosphingolipids are also efficiently recovered with the DRM,
suggesting that associations among components of these co-extracted
lipid components contribute to resistance against extraction by cold
nonionic detergent. DRMs are classically defined as microdomains or
membrane rafts. The properties and characteristics of DRMs in terms
of its existence on cells and its chemical behavior will be discussed in
the next section.
1.C: State of the membrane – ‘Raft Hypothesis’
1.C.i: Artificial membrane – description of ‘rafts’
Phase segregated domains have been demonstrated in artificial

membranes composed of ternary mixtures of lipids. These mixtures
exhibit three distinct phases depending on temperature and
5
Introduction
composition: gel (s o ), liquid ordered (l o ) and liquid disordered (l d ).
Above the chain melting temperature (T m ), the hydrocarbon chains of
lipids are floppy, disordered and loosely packed. This is known as
liquid (disordered) phase (l d ). The l d phase has short range positional
correlation. Below T m , lipids with saturated long acyl chains are tightly
packed and form a phase called the ‘gel phase’. The hydrocarbon
chains are oriented and ordered. The positional correlations in the
plane of the bilayer are long range. However, below T m , in the
presence of cholesterol, long saturated acyl-chains remain oriented but
the positional correlations are short range, like in a liquid. This is known
as liquid ordered phase (l o ). The diffusion coefficient of lipids in l o
phases is higher than in ‘gel-phase’, but lower in the l d phase. Since
rigid cholesterol molecules are inserted inside the lipid molecules (in
gel phase), the surface area per lipid molecule in the l o phase is larger
than in gel phase. However, above T m in the presence of cholesterol,
no macroscopic phase segregation was observed. But by
spectroscopic studies, such as nuclear magnetic resonance (NMR) and
electron spin resonance (ESR), the two fluid states (l o and l d ) were
shown to exist together (Sankaram and Thompson, 1990; Vist and
Davis, 1990). Below T m , where the gel phase is generally observed, is
replaced by l o phase in presence of high cholesterol (>20 mol%) and
the two fluid (liquid) phases (l o and l d ) can coexist (Brown and London,
2000). Experimentally, when the ternary mixture was brought down to
below T m of specific lipid species, these molecules form liquid ordered
phases (l o phase) in coexistence with disordered phases (l d ). These l o
phases coalesce into large scale domains which are resolvable by
optical microscopy. It is this l o phase that is thought to be relevant and
analogous to ‘lipid-rafts’ in biological systems.
6
Introduction
1.C.ii: Rafts in biological membrane redefined
Since, the l o domains exist as large-scale phase-segregated

domains, it was expected that they could be observed in biological
membranes using techniques such as fluorescence microscopy,
electron microscopy, optical tweezers, single molecules studies and
biochemical treatments (chemical cross-linking). However, in contrast
to the situation in artificial membrane, none of the above techniques
had been able to detect the presence of any large scale l o domain on
the native cell membrane (Mayor and Rao, 2004; Munro, 2003) . In this
scenario, several investigators tested the interaction of detergents with
membrane. This technique was used to assess the ‘fluidity’ of

biological membrane (Helenius and Simons, 1975). It was argued that
if biological membrane contains ‘gel-like’ l o patches, similar to artificial
membrane, these would be insoluble in cold-nonionic detergents (for
example Triton X-100). Consequently, it was found that when cell
membranes are extracted with cold (4°C) Triton X-100, a non-ionic
detergent, a small fraction of insoluble membrane residue consisting of
specific subsets of lipids and proteins – called ‘detergent resistant
membrane’ (DRM), is observed (Simons and Ikonen, 1997).
Compositionally, DRM has been correlated to the l o domain on the
model membrane (London and Brown, 2000). This membrane fraction
is enriched in cholesterol, sphingomyelin and many lipid-tethered
proteins such as non-receptor tyrosine kinase,
glycosylphosphatidylinositol anchored proteins (GPI-APs), etc (Simons
and Ikonen, 1997). However, careful biophysical studies in artificial
membrane have showed that Triton X-100 (TX-100) can induce
formation of ‘more ordered phase’ in model membranes from native
‘disordered’ phase (Heerklotz, 2002; Heerklotz et al., 2003).
Furthermore, it has been observed that TX-100 extracted (4°C) DRM
composition matches with the ‘l o ’ domain obtained in the ternary phase
7
Introduction
diagram at 37°C, but not with the composition of the l o phase at 4°C
(de Almeida et al., 2003). That means detergent extraction can also
change the composition of preexisting domain on any artificial
membrane. So, a priori existence of l o domain or ‘lipid-raft’ on native
cell membrane and its composition remains questionable (Mayor and
Rao, 2004).
1.D: Biophysical methods
The scale of lipid domains in cell membrane has remained

controversial; in a few experimental attempts, the size of lipid raft has
been estimated from <10nm to 700nm (Anderson and Jacobson,

2002). Various biophysical tools such as Förster’s Resonance Energy
Transfer (FRET), chemical cross linking, single particle tracking,
fluorescence correlation spectroscopy, laser trap have been used to
investigate the size of large scale organization of different molecules
(lipids, GPI-APs, toxins, trans-membrane proteins) at the cell surface.
However, all these measurements failed to provide consensus scale for
preexisting lipid domains at the cell surface.
GPI-APs have served as marker for rafts since they associate

with DRMs in a cholesterol sensitive fashion (Brown and Rose, 1992).
They also form cholesterol–sensitive nanoclusters (Varma and Mayor,
1998). These were characterized by measuring homo-FRET between
fluorescently labeled GPI-APs on the native cell membrane.
In contrast to hetero-FRET where FRET is monitored between

two different fluorophore, in the homo-FRET process, energy transfer
between two like fluorophores may be measured if they are in close
proximity (<10nm distance) (Varma and Mayor, 1998). This non-
invasive technique showed presence of sub-resolution (<70nm)
8
Introduction
clusters of GPI-APs at the live cell surface (Varma and Mayor, 1998).
These sub-resolution clusters are sensitive to cholesterol and
sphingolipid content in the membrane. However, lack of measurable
hetero-FRET between donor and acceptor fluorescent labeled GPI-APs
on the cell surface contradicted the possibility of nanoclusters in sub-
resolution domain (Kenworthy and Edidin, 1998). A resolution of this
controversy was obtained when Sharma et al measured the scale of
GPI-AP organization. They theoretically modeled the gradual change in
homo-FRET efficiency observed upon photobleaching of fluorophore-
labeled GPI-APs and it to obtain the size of GPI-AP structures giving
rise to the FRET signals (Sharma et al., 2004). Moreover, Sharma et al
provided an explanation for lack of detectable hetero-FRET between
donor-acceptor pair labeled GPI-AP molecules in nanoclusters by

calculating the theoretical values of average hetero-FRET between
them (Sharma et al., 2004).
Since last decade, there are lot of interesting aspects have been
brought up in the field of protein and lipid diffusion at the cell
membrane. Various research groups, using different techniques, have
proposed different models for plasma membrane diffusion. Amongst all
hypotheses, the hop-diffusion model proposed by Akihiro Kusumi
(Fujiwara et al., 2002) has significant evidence for hop diffusion. But in
the contrary, evidences from other laboratories, the hypothesis of hop
diffusion instead of typical Brownian diffusion is still not consensus and
leaves scope for a resolution.
There are groups of researchers who have also shown slowing

down of certain membrane proteins, which are associated to ‘lipid
rafts’. All these findings, however, also depend on the spatio-temporal
scale of measurements. Since it has been established that GPI-
anchored proteins, typical lipid raft associated protein, remain in
9
Introduction
cholesterol dependent sub-resolution clusters at the cell surface,
nobody has attempted to show its mobility and maintenance. Since,
identification of sub-resolution clusters is not straight-forward, it is very
difficult to study its dynamics. It is also important to realize the fact that
cluster mobility, molecular association kinetics and its dynamics is
inter-related. In this context, it is important to use the correct technique
and procedure to measure properties like mobility, kinetics and
maintenance of these clusters at the cell surface.
Previous studies in our laboratory, using fluorescence

anisotropy measurements on images as a measure of homo-FRET,
have shown that GPI-anchored proteins are present in sub-resolution
clusters at the surface of living cells (Varma and Mayor, 1998). Human
Folic acid receptor (FR) expressing CHO cells were labeled with
fluorescent analogues of folic acid Nα-pteroyl-Nε-(4’-
fluoresceinthiocarbamoyl)-L-lysine (PLF). The PLF bound FR were
excited with plane polarized light and wide-field emission fluorescence
anisotropy was measured in steady-state for whole cell. Typically, a
random equilibrated system should give rise to a density dependent
anisotropy profile (emission anisotropy decreases due to depolarization
caused by homo-FRET) as the average inter-fluorophore distance
decreases (one criterion for FRET to occur). The anisotropy profile
obtained from cells, containing different levels of protein on the cell
surface, was independent of protein concentration (direct measure
from total intensity). The partial photo-bleaching of fluorophores
increases the inter-fluorophore distance and hence rise of anisotropy
value (due to loss of homo-FRET originated depolarization). These
confirm existence of sub-resolution clusters. Since, it has also been
shown for a non-specific fluorescence protein (GFP) anchored by GPI
on the cell surface (GFP-GPI), this organization is truly based on the
anchor properties and independent of the protein characteristics
10
Introduction
(Figure 1.B). The anisotropy decay rate obtained from time-resolved
anisotropy measurement on surface of GFP-GPI expressing cell was
another crucial result to determine the properties of these clusters.
Essentially, by modeling the bleaching-profile of fluorophores attached
to the receptors, the characteristic of GPI-AP organization at the cell
surface was elucidated (Figure 1.C). This depicts that nano-clusters
consists 2-4 molecules in each cluster and 20-40% of such clusters are
present on cell surface (Sharma et al., 2004). Furthermore, high
resolution wide-field anisotropy imaging (work done by Sameera
Bilgrami in our laboratory) reveals that nano-clusters are distributed
heterogeneously on the cell surface.
Properties of GPI-AP organization till date can be summarized

into following points: a) GPI-APs remain in a small fraction of
nanoclusters, 20-40% of the total protein on the cell surface, consists
of 2-4 molecules per cluster. b) nanoclusters are cholesterol sensitive.
c) Nanoclusters are always maintained a particular concentration
irrespective of surface protein concentration. This feature does not
obey the ‘law of mass action’ – represents a non-equilibrium state. d)
Multiple GPI-AP can cohabit within a cluster and crosslinking with the
antibodies remodels the cluster composition. This means clusters are
not frozen and they do exchange molecules with monomers present at
the cell surface and hence new clusters are obviously made on the cell
surface.
This description provides an average picture of GPI-AP

organization at the cell surface at 20°C which lacks information about
the dynamics of the aggregation process or its spatial heterogeneity; it
also lacks an understanding of the non-equilibrium state of the whole
process.
11
Introduction
1.E: Objective of the thesis
The present status and understanding of properties of ‘lipid-rafts’

in the view of GPI-AP organization at the cell surface evokes two
obvious questions. Firstly, the nature of the steady state dynamics and
distribution of nanoclusters on the cell surface and secondly, the
underlying mechanism necessary for the maintenance of such unusual
organization. Therefore, I decided to address the following set of
questions and attempt to answer them by developing necessary tools:
1. The diffusivity of nanoclusters vs. monomers at the cell

surface.
2. The distribution of nanoclusters at the cell surface at high

resolution, which requires high resolution anisotropy
imaging.
3. The nature of the steady state dynamic properties of
nanoclusters on the cell surface – the formation and
fragmentation process.
4. Mechanism of maintenance of nanoclusters.
5. Perturbation of the nanocluster organization.
12
Introduction
1.F: References
Anderson, R.G., and K. Jacobson. 2002. A role for lipid shells in

targeting proteins to caveolae, rafts, and other lipid domains.
Science. 296:1821-5.
Brown, D.A., and E. London. 2000. Structure and function of
sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem.
275:17221-4.
Brown, D.A., and J.K. Rose. 1992. Sorting of GPI-anchored proteins to
glycolipid-enriched membrane subdomains during transport to
the apical cell surface. Cell. 68:533-44.
Chatterjee, S., E.R. Smith, K. Hanada, V.L. Stevens, and S. Mayor.

2001. GPI anchoring leads to sphingolipid-dependent retention
of endocytosed proteins in the recycling endosomal
compartment. EMBO J. 20:1583-1592.
de Almeida, R.F., A. Fedorov, and M. Prieto. 2003.
Sphingomyelin/phosphatidylcholine/cholesterol phase diagram:
boundaries and composition of lipid rafts. Biophys J. 85:2406-
16.
Frye, L.D., and M. Edidin. 1970. The rapid intermixing of cell surface
antigens after formation of mouse-human heterokaryons. J Cell
Sci. 7:319-35.
Fujiwara, T., K. Ritchie, H. Murakoshi, K. Jacobson, and A. Kusumi.
2002. Phospholipids undergo hop diffusion in
compartmentalized cell membrane. J Cell Biol. 157:1071-81.
Heerklotz, H. 2002. Triton promotes domain formation in lipid raft
mixtures. Biophys J. 83:2693-701.
Heerklotz, H., H. Szadkowska, T. Anderson, and J. Seelig. 2003. The
sensitivity of lipid domains to small perturbations demonstrated
by the effect of Triton. J Mol Biol. 329:793-9.
13
Introduction
Helenius, A., and K. Simons. 1975. Solubilization of membranes by
detergents. Biochim Biophys Acta. 415:29-79.
Kenworthy, A.K., and M. Edidin. 1998. Distribution of a
glycosylphosphatidylinositol-anchored protein at the apical
surface of MDCK cells examined at a resolution of <100 A using
imaging fluorescence resonance energy transfer. J Cell Biol.
142:69-84.
London, E., and D.A. Brown. 2000. Insolubility of lipids in triton X-100:
physical origin and relationship to sphingolipid/cholesterol
membrane domains (rafts). Biochim Biophys Acta. 1508:182-95.
Low, M.G., and J.B. Finean. 1977. Release of alkaline phosphatase
from membranes by a phosphatidylinositol-specific
phospholipase C. Biochem J. 167:281-4.

Mayor, S., and M. Rao. 2004. Rafts: scale-dependent, active lipid
organization at the cell surface. Traffic. 5:231-40.
Mayor, S., and H. Riezman. 2004. Sorting GPI-anchored proteins. Nat
Rev Mol Cell Biol. 5:110-20.
Munro, S. 2003. Lipid rafts: elusive or illusive? Cell. 115:377-88.
Paulick, M.G., and C.R. Bertozzi. 2008. The
glycosylphosphatidylinositol anchor: a complex membrane-
anchoring structure for proteins. Biochemistry. 47:6991-7000.
Sankaram, M.B., and T.E. Thompson. 1990. Modulation of
phospholipid acyl chain order by cholesterol. A solid-state 2H
nuclear magnetic resonance study. Biochemistry. 29:10676-84.
Sharma, P., R. Varma, R.C. Sarasij, Ira, K. Gousset, G.
Krishnamoorthy, M. Rao, and S. Mayor. 2004. Nanoscale
organization of multiple GPI-anchored proteins in living cell
membranes. Cell. 116:577-89.
Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes.
Nature. 387:569-72.
14
Introduction
Singer, S.J., and G.L. Nicolson. 1972. The fluid mosaic model of the
structure of cell membranes. Science. 175:720-31.
Tanaka, S., Y. Maeda, Y. Tashima, and T. Kinoshita. 2004. Inositol
deacylation of glycosylphosphatidylinositol-anchored proteins is
mediated by mammalian PGAP1 and yeast Bst1p. J Biol Chem.
279:14256-63.
Varma, R., and S. Mayor. 1998. GPI-anchored proteins are organized
in submicron domains at the cell surface. Nature. 394:798-801.
Vist, M.R., and J.H. Davis. 1990. Phase equilibria of
cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear
magnetic resonance and differential scanning calorimetry.
Biochemistry. 29:451-64.
15
Introduction
Figure 1.A GPI-anchor and anchored proteins
i. Core structure of GPI-anchor - The membrane anchoring is

done by two fatty acyl chains linked to a glycerol backbone. The third
hydroxyl of the glycerol esterified to a phospho-inositol group that is
further linked to an oligosaccharide consisting of glucosamine and
mannose residues and a terminal phosphoethanolamine that is linked
to the carboxy-terminal cysteine of the protein. Taken from:
Sabharanjak S,et al. Adv Drug Deliv Rev 2004 Apr 29;56(8):1099-109.
ii. Cartoon representation of membrane anchored folate receptor
labeled with fluorescence analogue of folic acid (PLF) and GFP-GPI, a
model protein synthesized inside the cell. Taken from: Sharma P, et
al.Cell. 2004 Feb 20;116(4):577-89.
16
Introduction
Figure 1.B Concentration independent anisotropy at cell surface
Mean fluorescence intensity and anisotropy is plotted in the

graph for GFP-GPI expressing CHO cells grown under normal
condition (black solid circle) and cholesterol depleted condition (red
solid circle). The organization of GPI-anchored protein is independent
of protein concentration at the cell surface. This organization is also
sensitive to level of cholesterol at cell surface. Taken from: Sharma P,
et al.Cell. 2004 Feb 20;116(4):577-89.
17
Introduction
Figure 1.C Pictorial representation of GPI-anchored protein
organization
The picture of GPI-AP organization obtained from the theoretical

estimation of the cluster to monomer ratio at the cell surface. This was
generated by fitting the bleaching profile obtained from the
experimental data of protein organized at the membrane. GPI-APs are
distributed as 20-40% of clusters (with size of ≥ 2 proteins) and
monomeric proteins. The inter protein distances within the cluster is of
the order of R0 , Forster’s radius (scale bar). Taken from: Sharma P, et
al.Cell. 2004 Feb 20;116(4):577-89.

18
Chapter 2
Materials and methods
2.A: Cell lines
Chinese Hamster Ovarian (CHO) cells stably transfected with

two different types of GPI-anchored proteins were used according to
the requirement of experiments. Details of these cell lines are given in
the table below:
Cell Source Description Reference

lines
1 IA2.2F S. Mayor CHO cells (TRVB), devoid of Sabharanjak

transferring receptor (Tfr), were et. al. 2002
stably transfected with human
Tfr (geneticin selection) and
GPI-anchored protein – the
human folate receptor (FR-
GPI) (hygromycin selection).
2 GG8 S. Mayor CHO cells (TRVB), devoid of Sabharanjak

transferring receptor (Tfr), were et. al. 2002
stably transfected with human
Tfr (geneticin selection) and
GFP-GPI (hygromycin
selection).
19
Materials and Methods
2.B: Cell culture media and imaging buffer
Ham’s F12 medium (HF12) from HiMedia (Bombay, India)

supplemented with 10% fetal bovine serum (FBS) from GIBCO
(Carshland, CA, USA), appropriate selection antibiotic and combination
of penicillin and streptamycin as antibacterial agent (Chadda et al.,
2007). FR-GPI cells were grown in folic acid free HF12 medium
supplemented with dialysed serum. Appropriate concentration of
fluorescent labels were prepared with either growth medium or medium
1 (150nM NaCl, 5mM KCl, 1mM CaCl 2 , 1mM MgCl 2 , 20mM HEPES,
pH7.4). Cholesterol depletion reagents were also made in medium 1.
Cells were washed with and imaging was performed in presence of

medium 1 after labeling and / or necessary treatments. For
experiments performed at 37°C 1mg/ml glucose and BSA (bovine
serum albumin) was added to medium 1.
2.C: Fluorescence labeling on cell surface
Folic acid expressing cells were visualized using three different

fluorescence analogues of folic acid PLF (Nα-pteroyl-Nε-(4’-
fluoresceinthiocarbamoyl)-L-lysine, PLBTMR (Nα-pteroyl-Nε-(4’-
BodipyTMR)-L-lysine) or PLR (Nα-pteroyl-Nε-(4’-lissamine rhodamine
thiocarbamoyl)-L-lysine). Fluorescence probe stocks were maintained
at -20°C (described in (Sabharanjak et al., 2002)). PLF, PLBTMR and
PLR saturation binding concentrations for cells were obtained 80nM,
40nM and 400nM respectively, of which PLF saturation profile is shown
in Figure 2.A. Surface labeling was performed by incubating cells with
PLF, PLBTMR or PLR on ice for 1 hour. Excess fluorophore was
removed and cells were washed with ice cold medium 1. GG8 cells
20
were treated with 50µM cycloheximide to stop protein expression for 3
hours at 37°C to obtain surface fluorescence only. In Figure 2B, GG8
cell are shown without (i) or with (ii) cycloheximide treatment.
2.D: Cholesterol depletion procedures
Saponin treatment: Labeled cells with appropriate fluorophores

(according to the requirement) were treated with saponin. Pre-chilled
0.3% saponin in medium 1 was used on cells on ice for 30 minutes
(Cerneus et al., 1993).
Cyclodextrin treatment: 10 mM methyl-β-cyclodextrin (mβCD)
solution in medium 1 was prepared at 37°C (Yancey et al., 1996). It

was used on cells according to the protocol applicable to specific
experiment described in chapters later on.
2.E: Actin or myosin perturbing procedures
Two main methods for treatment of cells with actin or myosin

perturbing agents.
To generate blebs:
Cells were incubated at 37°C in growth medium (HAM’s F12) ,
containing 25µM latrunculin or 14µM jasplakinolide for 30 minutes,
either after cycloheximide treatment of GG8 cells or before labeling of
FR-GPI on IA2-2f cells with PLF. Stable membrane deformations and
blebs were formed after 30 minutes of treatment. Time resolved
anisotropy and fluorescence lifetime measurements on blebs were on
these blebs were conducted as described in the next chapter.
During the microphotolysis-type FIAT experiments:
21
For microphotolysis type of experiments, 1µl of latrunculin or jas

plakinolide at a concentration of 6mM or 5mM respectively was added
to 1ml of M1 and 1mg/ml glucose and BSA on cells in a 35mm dish
kept on the microscope stage maintained at 37°C. Time of treatment
optimization and photon collection is as described in each experiment.
2.F: Wide field microscopy
TE-2000 or TE-300 from Nikon (Japan) microscope was used

for both wide field fluorescence microscopy and wide field anisotropy
imaging. Briefly, the microscope was equipped with a mercury arc lamp
as an excitation light source and excitation filter wheel for choosing the
excitation wavelength from Shutter instruments (Novato, CA, USA).
Appropriate dichroics and matching excitation and emission filters were
used for imaging. For simultaneous dual color imaging, dual pass
excitation filters were used for simultaneous excitation in two
wavelengths. A secondary beamsplitter was used after the sideport to
separate the emission light intensities for the shorter and longer
wavelengths. In case of anisotropy imaging a polarizing beam splitter
was placed. Images were collected with two 16-bit cooled back
illuminated frame transfer EMCCDs set at 1Mhz transfer rates for
normal gain settings. The instruments were controlled by Metamorph
software.
For 20X steady state anisotropy measurements:
Sub-resolution bead images (Figure 2.C.i-iii) were used to align

two cameras with respect to each other by adjusting the polarizing
beam splitter and position of two cameras. Field illumination was also
optimized to deliver a flat field (Figure 2.C.iv). A set of solution
experiments was performed to test whether the microscope was
22
capable of detecting changes in anisotropy. Rhodamine 6 G dye was
dissolved in varying glycerol concentrations to obtain solutions of
different viscosity. Since, fluorescence emission anisotropy reports the
extent of rotational motion, the steady-state anisotropy value will
increase as the molecular rotational motion is restricted (Figure 2.D.i)
when the concentration of glycerol increases. To measure the extent of
depolarization of emission anisotropy due to FRET (Figure 2.D.ii), the
concentration of Rhodamine 6G dye in 70% glycerol solution (70%
glycerol concentration solution was used to hinder the rotation of small
molecule to some extent that has a measurable anisotropy value) was
increased to an extent where average distance between molecules
was less than Forster distance and hence FRET expected in those
concentrations. Furthermore, to verify the accuracy of the microscope a

set standard solution measurements, where anisotropy of fluorescence
emission of Rhodamine dye in 70% glycerol and GFP in PBS
(phosphate buffer saline) were compared before each experiment.
Solution or cell images were acquired simultaneously for parallel

and perpendicular camera by the Metamorph software and split to
obtain separate parallel and perpendicular images using the ‘Split view’
function from Metamorph software. Glycerol or M1 image intensities
were recorded as blanks and were subtracted from the corresponding
solution or cell images. Regions were cut around solution or cells,
intensity logged onto excel worksheet and anisotropy ( r ) calculated as
I − I ⊥
r=
I + 2 I ⊥
where, I  and I ⊥ are the calculated intensities of fluorescence
emission with polarization parallel and perpendicular to the

excitation polarization, respectively, I= I  + 2 I ⊥ is the total
23
fluorescence emission intensity, and r is the fluorescence
anisotropy.
The perpendicular image is always corrected for the instrument

bias, called G factor. This was calculated as a ratio Ipa/Ipe obtained
from a concentrated fluorescein solution in water that gave intensity
values, ten times those obtained on cells. It was determined separately
that G factor values remain constant with fluorescein concentration.
Intensity bins of 200-500 units were used to calculate average
anisotropy, and the standard deviation (SD) in that bin. Two dishes per
treatment were imaged, and anisotropy with standard error of the mean
was calculated for each of the intensity bin.
2.G: Confocal microscopy
Zeiss LSM 510 meta and LSM 5 LIVE (Germany) microscope

was used for confocal and multiphoton fluorescence anisotropy
experiments. Details of instrumentation and analytical methods for
fluorescence anisotropy, time resolved fluorescence experiments and
are elaborated in the next chapter.
24
2.H: Refrerences
Cerneus, D.P., E. Ueffing, G. Posthuma, G.J. Strous, and A. van der

Ende. 1993. Detergent insolubility of alkaline phosphatase
during biosynthetic transport and endocytosis. Role of
cholesterol. J Biol Chem. 268:3150-5.
Chadda, R., M. Howes, S. Plowman, J. Hancock, R. Parton, and S.
Mayor. 2007. Cholesterol sensitive Cdc42-activation regulates
actin polymerization for endocytosis via the GEEC pathway.
Traffic. in press.
Sabharanjak, S., P. Sharma, R.G. Parton, and S. Mayor. 2002. GPI-
anchored proteins are delivered to recycling endosomes via a

distinct cdc42-regulated, clathrin-independent pinocytic
pathway. Dev Cell. 2:411-23.
Yancey, P.G., W.V. Rodrigueza, E.P. Kilsdonk, G.W. Stoudt, W.J.
Johnson, M.C. Phillips, and G.H. Rothblat. 1996. Cellular
cholesterol efflux mediated by cyclodextrins. Demonstration Of
kinetic pools and mechanism of efflux. J Biol Chem. 271:16026-
34.
25
Figure 2.A Saturation binding assay for PLF probe
Example of how saturation binding was obtained for each probe

that binds to FR-GPI expressing CHO cells (IA2.2F). IA2.2F cells
grown in folate free HF12 medium were labeled with varying
concentration of PLF o ice for 1hr. Average fluorescence intensity from
150 cells (integrated intensity was calculated by drawing outline for
each cell in a field) was plotted for each concentration of probe. The
graph showed that the average intensity did not increase as labeling
concentration increased from 80nM to 160nM. Therefore, the
saturation concentration of PLF probe is between 80-160nM.
26
Figure 2.B Images of GG8 cells
Images of GG8 cell in a confocal microscope before (i) and after

(ii) cycloheximide treatment for 3 hrs at 37°C, showing that
cycloheximide treatment effectively reduces intracellular GFP-
fluorescence. Scale bar 10µm.
27
Figure 2.C Wide-field illumination profile
200nm beads imaged in wide-field two camera setup (i, parallel

camera) and multiphoton (ii, perpendicular camera) was overlapping
with each other (iii, yellow). Uniform fluorescence intensity profile (flat-
field) obtained when fluorescein solution was imaged with both camera
(image in iv, is obtained in parallel camera). Both are important for
anisotropy measurements.
28
Figure 2.D Measuring small systematic changes of anisotropy value
i. Anisotropy values obtained from Rhodamine 6G (10µM; at this

concentration there is no FRET) were plotted with varying glycerol
concentration. A systematic increase in anisotropy value was observed
which follows Perin’s equation. ii. Anisotropy values were obtained
from varying concentration of Rhodamine 6G in 70% glycerol (at this
concentration small molecule like Rhodamine 6G has measurable
anisotropy value) concentration. There is systematic decrease of
anisotropy due to homo-FRET.
29
Chapter 3
Microscopes, instrumentation and analytical

methods for FRET-based measurements on cells
3.A: Introduction
Fluorescence resonance energy transfer (FRET) is a method to

detect proximity between fluorophores at 1-10nm scales. Molecular
interactions, monitored by FRET-based spectroscopic measurement,
can also be implemented in microscope to determine the sizes of
optically unresolved structures at the cell surface. This has been

efficiently employed in our laboratory (Rao and Mayor, 2005; Sharma
et al., 2004; Varma and Mayor, 1998). Previously, in our laboratory,
homo-FRET measurements were performed on a wide-field
fluorescence microscope with a poorly resolved optics where 20X -
0.75 numerical aperture (NA) objective was used to obtain average
information of GPI-anchored protein organization on the cell surface.
To obtain minute details of spatial distribution and formation kinetics of
this organization, high-resolution (both lateral and axial) FRET imaging
was required. In addition, steady-state FRET measurements are often
unable to reveal details of molecular organization. In that case, time
resolved fluorescence measurements are necessary to uncouple the
intertwined contributions of molecular rotation and FRET-contributions
to the steady state measurements.
3.A.i: Anisotropy-based FRET measurements
Fluorescence anisotropy is a property of rotation of fluorophore

or its emission dipole moments. The selection of fluorophore during its
30
FRET Microscopy: Theory and Tools
excitation completely depends on the polarization property of the
exciting light. Homo-FRET is measured using the polarization property
of light. Polarization may be altered due to many physical parameters,
for example, magnetic field, physico-chemical properties and molecular
structure of the conducting medium. After polarized excitation of
fluorophores, its emission may be depolarized due to i) very fast
molecular rotation, ii) a marked angle between emission and excitation
dipoles, or ii) energy transfer to randomly oriented neighbouring
fluorophores (Lakowicz, 1999). In steady-state detection system, a low
anisotropy value can be obtained; the exact source of depolarization
may remain obscure.
Anisotropy of the fluorescence detected also depends on the

microscopic system used. In context of an epi-fluorescence
microscope, specifically, polarization of excitation or emitted light
depends on its interaction with optical components such as dichroic,
optical filter and objective. High NA objectives bend light in steep angle
which causes depolarization of polarized light. In wide-field setup,
usage of high NA (numerical aperture; above 1.2) objectives leads to a
fully depolarized fluorescence emission (of the order of ~0.3) due its
optics. So, any spatial or temporal change in anisotropy, utilized as a
reporter of FRET on the cell surface (Rao and Mayor, 2005), cannot be
distinguished. To retain the anisotropy in the fluorescence emission
with a high NA microscopic system, the dynamic range of anisotropy
detection must be increased. This can efficiently be done in two ways:
i) confocal detection of emitted fluorescence that will not allow
detection of the out-off focus depolarized photons and select relatively
polarized focal plane fluorescence; ii) a further narrow photo-selectivity
by engaging two-photon excitation of fluorophores. A pulsed two-
photon excitation also allows us to monitor time-resolved fluorescence
31
measurements with the help of time correlated single photon counting
(TCSPC) method.
3.A.ii: Fluorescence lifetime based FRET measurements
Alteration in the fluorescence lifetime provides direct a

quantitative measure of hetero-FRET, a process when resonance
energy transfer occurs between two different types of fluorophore
having significant spectral overlap. In presence of an acceptor
fluorophore in proximity, donor fluorophore transfer its energy to the
acceptor molecule in non-radiative fashion. In this process the average
lifetime of the donor fluorescence decreases (Lakowicz, 1999). With
the help of TCSPC method, donor fluorescence lifetime can be

measured and the true efficiency of FRET can be calculated.
This is a complementary method also used to determine GPI-AP

organization on the cell surface. According to the theoretical modeling
(Sharma et al., 2004), the size and molecular ratio in a molecular
organization can be predicted from hetero-FRET studies where donor
to acceptor fluorescence ratio is varied. Hetero-FRET measurements
are also quite useful for fluorophores that remain aligned in biological
samples where anisotropy measurement fails.
3.B: Multiphoton laser scanning confocal microscope
3.B.i: Microscope setup
1. Zeiss LSM 510 META confocal microscope. Zeiss

software was used for image acquisition, specifying
region of interest (ROI), laser beam steering and parking.
32
Acousto-optic tunable filter (AOTF) is the part of this
microscope for laser power control (Figure 3.A).
2. Single photon lasers: Ar ion laser (LASOS) emitting

475nm, 488nm, 405nm, 514nm; He-Ni lasers (LASOS)
for 532nm and 633nm excitation wavelength respectively.
3. Tsunami Titanium:Sapphire (Ti:Sa), a tunable (700nm-

950nm) multiphoton femto-second pulsed (80 MHz) laser
from Spectra physics was coupled to the microscope.
4. Objectives: 63X (1.45NA), 20X (0.75NA), 10X (0.3NA),

20X (0.5NA), 40X (1.45NA).
5. Two Hamamatsu Photomultiplier tubes (PMTs) at

descanned side and a meta detector with a series of
seven PMTs for spectral imaging.
6. Two Hamamatsu R3809U multi-channel plate

photomultiplier tubes (PMTs; Hamamatsu Photonics) at
the non-descanned detection path.
7. Polarizing beam splitter (PBS) cube (Melles Griot) at the

non-descanned detection path for collection of parallel
and perpendicular emitted photons in case of anisotropy
measurements.
8. Appropriate set of dichroic beam-splitters, emission filters

were chosen and pre-installed before the pinhole, inside
the microscope confocal scan-head.
33
9. Time correlated single photon counting card (TCSPC 830
card) was installed from Becker & Hickl, for time resolved
fluorescence data collection that operates in a stop–start
configuration.
3.B.ii: Advantages of multiphoton excitation
1. Fluorescence excitation happens within a confocal

volume (Volkmer et al., 2000; Zipfel et al., 2003) (Figure
3.B). Two-photon excitation was confirmed from log-plot
of power of excitation vs. fluorescence emission from a
fluorescein solution that has a slope of two (Figure 3.C).
2. Narrow photo-selection with respect to single photon

excitation offers higher dynamic range in anisotropy
(Figure 3.D).
3. High NA objectives offer better resolution, higher

convergence of multiphoton laser but result in more
effective depolarization of the emitted fluorescence due to
the high NA.
4. Less bleaching of fluorophores which are out of focus of

the objective.
5. Data can be collected from an isolated point in cell such

as sub-cellular structures, membrane deformities,
endosomes etc.
6. Since multiphoton excitation is pulsed, it can also be used

for time resolved fluorescence studies.
34
3.B.iii: Time-resolved fluorescence measurements from

living cells using TCSPC 830 card.
Fluorescence lifetime and time-resolved anisotropy

measurement experiments were performed on the TCSPC 830 card
which is connected to the MCP-PMTs attached with above mentioned
microscope (Figure 3.A). Intensity decay profiles (for lifetime or time-
resolved anisotropy) can be obtained by TCSPC method without
accounting all photons arrived at the MCP-PMTs (Becker, 2005;
Lakowicz, 1999). Excitation events occur when laser pulses hit the
sample. Part of the excitation pulse is sent to reference PMT which
generate a start signal. This signal starts time-to-amplitude converter

(TAC), which generates linear ramp voltage unless another pulse from
record PMT (generated by photon from the fluorescence event) stops
it. The TCSPC measurements relies on the concept that the probability
distribution for the emission of a single photon after an excitation yields
the actual intensity versus time distribution of all the photons emitted as
a result of an excitation (Figure 3.E). By multiple sampling of the time
interval between excitation and the arrival of single photon from a very
huge number of excitation events, the card constructs this probability
histogram of counts versus time channel (Figure 3.E). This represents
the actual intensity versus time distribution of the sample. The
description of the setup used for TCSPC measurements is as follows:
1. Ti:Sapphire Tsunami laser was used as the multiphoton

pulsed excitation light source. It is a tunable laser whose
wavelength ranges from 720-1080nm. It was tuned and
mode locked at 790nm and 920nm wavelength for
excitation of GFP and fluorescein respectively. The
35
repletion rate of the pulse laser is 80MHz which means
pulse appears in every 12.2ns.
2. A small fraction (1percent) of the laser was deflected to

the reference photodiode which provide the reference
time for incoming laser pulse to the TCSPC 830 card.
3. AOM was used to control the laser power. The laser is

vertically polarized. Reverse dichroic (650nm long pass)
allows IR laser to the sample and fluorescence (less than
650nm wavelength) deflect the photons towards non-
descanned side of the microscope. Emitted photons are
selected with a band-pass filter (IR blocked) according to

their emission wavelength range. Photons are then split,
using a polarizing beam splitter, according to their
polarization and recorded simultaneously in two MCP-
PMTs used for detection. In case of fluorescence lifetime
detection, all fluorescence photons are recorded with a
polarizer in the emission path at 54.7° in a single MCP-
PMT. Zeiss 20X 0.7NA and 63X 1.45NA objectives was
used according the experimental protocol.
4. Due to the random gain mechanisms (secondary

electrons generated by the PMT have wide range of
trajectories and velocities) jitter is inherent in single
photon pulses. The temporal location of incoming single
photon pulses is made more accurate by feeding it to
constant fraction discriminator (CFD). CFDs generate
zero cross-over point by adding input pulse to delayd and
inverted input pulse. The temporal position of zero cross
point is independent of amplitude. Moreover, CFD rejects
36
input pulses smaller than detector threshold and larger
than selected amplitude window, leading to significant
improvement of signal to noise. The pulses from start and
record PMTs are individually passed through CFDs
before they reach time-to-amplitude converter (TAC).
5. TAC is the key of single photon counting method. It

measures the time difference between excitation pulse
and the detected photon resulting from fluorescence. A
start pulse received by the TAC generates linear ramp
voltage until signal from stop pulse from the fluorescence
photon detector arrives. The output voltage has linear
dependence on the time difference between arrival of the

start pulse and stop pulse. In ‘start-stop’ mode the laser
pulse can start the TAC and fluorescence photons can
stop it. Alternatively, in a reversed ‘start-stop’ mode,
fluorescence photons can act as a start signal and the
next laser pulse to stop it. Since the ‘start-stop’ mode
results in large dead time for TAC (~99% of the time start
pulse is not followed by any fluorescence emission
photons, resulted aborted events), it can only handle
slower repetition rates. In this set up reverse ‘start-stop’
mode is implemented since the laser repetition rate is 80
MHz.
6. Analogue pulse from TAC is fed into multi channel pulse

height analyzer (MCPHA). Analogue to digital conversion
is done by this MCPHA, where amplitude of TAC pulse
and registers the event into designated time channel.
Multiple such events are counted and histogram of counts
versus time is constructed. The measurements are
37
usually continued upto 1,000-10,000 counts (at the peak
time) in each side (parallel and perpendicular channel).
7. After arrival of the first fluorescence photon, TAC can be

started to measure the time until the stop-pulse arrives
from the laser. During this time interval subsequent
photons are lost and that is known as dead-time of the
system (typically in µs order). Hence, if the probability of
photon arrival after each excitation pulse is <1, only early
photons would be detected by TAC leading to
underestimation in the decay time (also known as pile
up). These problems can be avoided by adjusting the
count rate such that the probability of detecting single

photon after excitation of laser pulse is <0.01 (in this
condition probability of detecting 2 photons would be
<(0.01)2, which is less than 1% single photon probability).
Therefore, the fluorescence photon count rate of
detection by MCP-PMT (r CR ) must be adjusted by the
following criterion by controlling the laser power or the
density of the fluorophore in the sample:
r CR < 0.01 r L
where, r L is the repetition rate of the laser.
8. That means, since laser repetition rate is 80 MHz,

fluorescence photon count rate must be less than 800
KHz.
9. The 12.5ns time period between two laser pulses is

divided into 1024 time channels which allow us to sample
38
intensity decay at the time resolution of 12.2ps per time
channel.
10. Dried colloidal gold particle, which emits instantaneous

photons of half of the wavelength of the excitation (a
phenomenon known as hyper-Rayleigh scattering), was
used collect the instrument response function (IRF)
(Figure 3.F). Scattered photons were collected in two
PMTs simultaneously with the same configuration. The
measured full width at half maximum was 60ps for this
system. This IRF was then used to analyze flurorescence
decays.
3.B.iv: Time-resolved fluorescence studies on live cells
1. Before any measurement on cells or solutions,

multiphoton laser was always aligned with respect to the
pre-aligned single photon laser installed within the
microscope. Both, field uniformity by acquiring image of
uniform density fluorescein solution and sub-resolution
beads distributed on a slide were used for alignment of
the multiphoton laser (Figure 3.G).
2. Before cellular measurements a standard fluorescent

dye, fluorescein in water at pH11, was always taken as
control to verify optimal properties for time-resolved
fluorescence measurements. The lifetime of fluorescein
at pH11 was ~4ns (Figure 3.H.i), which is similar to that of
reported in literature. Correlation time for a 500Da
molecule in water (such as fluorescein) is ~100ps. Hence,
this fluorophore having fluorescence lifetime greater than
39
1ns would undergo complete depolarization (Figure
3.H.ii). Anisotropy of fluorescein at pH11 in water rapidly
decayed to zero (Figure 3.H) and was also used to
estimate the G Factor by adjusting the factor multiplied to
the perpendicular photons for correction of the bias
present in the collection optics of the microscope during
fluorescence anisotropy measurements.
3. Time resolved anisotropy (TRA) decay was taken from

GFP in PBS (aqueous solution) at pH7.0 as reference for
anisotropy measurements. The correlation obtained was
similar to that reported the existing literature. Since all
measurements were performed on cells expressing GFP,

r 0 was used (with a very narrow window) for fitting data
from cell that was estimated from the multiple fitting trials
of GFP in solution.
4. Cells expressing various GFP-constructs were used for

TRA measurements. For multiphoton excitation of GFP or
fluorescein in cells, we used 920-nm excitation
wavelength. At this wavelength, the two-photon
absorption cross section for GFP is higher, enabling
lower laser excitation power, and auto-fluorescence
signals are minimized. The beam was ‘‘parked’’ at a
single point using routines available in the Zeiss software.
The parked beam was placed at the center of the field to
maintain uniformity of G-Factor, and photons were
collected for 30–50 s. Laser power was kept low such
that photons were collected at a maximum rate of 0.1
MHz and minimum rate of 10 KHz to ensure that TCSPC
conditions were strictly met and to maintain background
40
noise <1% of the signal. Since, the low laser power, less
than 10% bleaching was observed during a
measurement.
5. Fluorescence lifetime was obtained from flat regions of

cells, labeled with only donor or donor-acceptor with
varying ratios. Due to low photon counts in individual
pixels, satisfactory fluorescence decay curves were
constructed by scanning the laser over a small ~100x100
pixel area on the cell surface. Measurements on Blebs
were made by collecting photon statistics using a parked
beam located on the bleb. Fluorescence photons were
recorded by selecting photons at emission side

polarization at the magic angle, 54.7° to the excitation
beam. The angle of the polarizer was optimized by
sending low power polarized light in the emission path in
presence of the polarizer at 54.7°and estimating the
anisotropy value to be zero from the parallel and
perpendicular photons obtained at both the detectors.
3.B.v: Time-resolved fluorescence decay analysis
Fluorescence intensity decays:
Fluorescence intensity decays taken at 54.7° angle are analyzed

as sum of discrete exponentials (Grinvald and Steinberg, 1974; Krishna
et al., 2001; Krishnamoorthy et al., 1987; Lakowicz, 1999).
Experimental decay data, F(t) represents a convolution of instrument
response function R(t) with an intensity decay function I(t). It can be
represented as follows
41
t
F (t ) = ∫ R( s + δ ) I (t − s )ds
0
where, δ is shift parameter arising due to time shift between

measurement of instrument response and fluorescence photon.
Intensity decay function I(t) is sum of exponential and can be described
as follows:
n
I (t ) = ∑ α i e − t /τ i
i =1
where, τ i is the i th lifetime;
α i is the pre-exponential factor (amplitude) of i th lifetime

Since the system allows only 12.5ns time of decay, fluorophores

having lifetime more than 2ns would not decay fully. In such cases, the
residual photon out of fluorescence decay would add up at the
beginning of the decay, a process known as ‘roll-over’. Decays were
then fitted by an iterative reconvolution procedure using a Levenberg-
Marquardt minimization algorithm where roll-over effect has been
incorporated. The goodness of the fit was judged when three criteria
were met: a) reduced χ 2 was less than 1.2, b) residuals were evenly
distributed across the full extent of the data, and c) visual inspection
ensured that the fit accurately described the decay profile.
Time resolved anisotropy decays:
Polarized emission decays are analyzed to obtain rotational

parameters (Gryczynski et al., 1991; Krishna et al., 2001; Lakowicz,
1999). Parallel and perpendicular decays are described by following
equations:
42
1
=I I (t )[1 + 2r (t )]
3
1
=I⊥ I (t )[1 − r (t )]
3
where, r (t ) is anisotropy as function of time

and I (t ) is total intensity as function of time
r (t ) can be represented by the following equation:
I  (t ) − I ⊥ (t )
r (t ) =
I  (t ) + 2 I ⊥ (t )
The experimental data is fit to sum of two exponentials to obtain

rotational correlation time and respective amplitudes using following
equation (Krishna et al., 2001; Lakshmikanth and Krishnamoorthy,
1999; Swaminathan et al., 1996):
m
(r0 − r∞ )∑ β j e
( − t /τ rj )
r (t ) = +r∞
j =1
where, τ rj = j th roational correlation time
β j = pre-exponential (amplitude) for corresponding τ rj
r0 = initial anisotropy at t = 0
r∞ =residual anisotropy at infinite time
This equation reduces to the following form for:

a. mono-exponential decay (typical spherical probe that rotates
freely in the solution)
r (t ) = r0 e( − t /τ r )
43
b. bi-exponential decay
=r (t ) r0  β1e( − t /τ r 1 ) + β 2 e( − t /τ r 2 ) 
Rotational diffusion and homoFRET each result in exponential

decays in anisotropy which, for large proteins, occur on very different
time scales: homoFRET results in a rapid anisotropy decay, and
rotational diffusion results in a slower decay. Two decay models (as
explained earlier) for each empirical anisotropy decay: (1) a single
exponential decay and (2) the sum of two exponential decays were
used. Decay profiles, obtained from cells, describe anisotropy decay
through only a single process, presumably rotational diffusion. For a

second class, those were not fit well by a single exponent, but the
addition of a second exponent resulted in a good fit. These profiles
describe decay in anisotropy through two exponential processes, both
homo-FRET and rotational diffusion. Unlike fluorescence lifetime
decay, both parallel and perpendicular decays were analyzed
separately by an iterative reconvolution procedure using a Levenberg-
Marquardt minimization algorithm where roll-over effect was
incorporated (Krishnamoorthy et al., 1987). As previously described,
the goodness of the fit was judged when three criteria were met: a)
reduced χ 2 was less than 1.2, b) residuals were evenly distributed
across the full extent of the data, and c) visual inspection ensured that
the fit accurately described the decay profile.
44
3.B.vi: Steady-state anisotropy measurements using TCSPC

830 card.
Steady-state imaging was performed on Zeiss LSM 510 META

using multiphoton excitation. Scanner was set for a pixel residence
time of 102 µs/pixel. Imaging was performed using two MCP-PMTs
simultaneously in single photon counting mode on TCSPC 830 card.
The time resolution in the Becker and Hickl card was set to one so that
all the photons were binned into a single time channel. A full image
(512X512 pixel) was collected over 62 seconds.
3.B.vii: Steady-state anisotropy analysis
Steady-state anisotropy was calculated from steady-state

parallel- and perpendicular-polarization images. To account for
differences in the optical paths traversed by the perpendicular and
parallel emissions, a G-Factor correction was applied to the data as
follows. Parallel and perpendicular steady state emission images from
a fluorescein sample were collected. Because fluorescein tumbles
rapidly relative to the time scale of image acquisition, fluorescein emits
identically in both polarizations which is detected pixel-by-pixel readout
in two CCDs. G-Factor image from the parallel and perpendicular
fluorescein emission images were calculated, and perpendicular
images from subsequent experiments were multiplied by this G-Factor
image to apply the appropriate correction. Since pixel values in the
parallel and perpendicular images exhibit Poisson photon noise, the
images were averaged to reduce artifacts arising from dividing signals
containing noise and also to increase the signal-to-noise ratio. Three
pixel nearest neighbor averaging for parallel and perpendicular
fluorescein images were performed and then divided the averaged
images pixel by pixel to create G-Factor image. A new G-Factor image
45
was created for each day of experiments. Experimentally obtained
perpendicular image was multiplied with the G-Factor image. Specific
cases, using Metamorph software, regions of interest were manually
selected in the parallel image and were transferred to the perpendicular
image. The mean perpendicular- and parallel-polarization emission
intensities were calculated for each region, and from these, the steady-
state fluorescence anisotropy and fluorescence emission intensity were
calculated using the relations:
I − I ⊥
r=
I + 2 I ⊥
where, I  and I ⊥ are the calculated intensities of fluorescence
emission with polarization parallel and perpendicular to the

excitation polarization, respectively, I= I  + 2 I ⊥ is the total
fluorescence emission intensity, and r is the fluorescence

anisotropy.
3.C: Line-scanning confocal microscope
3.C.i: Microscope setup
1. Customized Zeiss 5 LIVE confocal microscope (Figure

3.I). Here the laser passes through a cylindrical optics by
which the laser beam is spread over a line. This line of
excitation light is then reflected by a one-dimensional
mirror named ‘achrogate mirror’ to the objective placed in
the optical path. Post excitation, the fluorescence
emission from the sample goes to the dichroic mirror with
little regard to the thin achrogate mirror in its path.
46
Instead of pinholes for confocality, slits are used to allow
only the fluorescence from focus of the line illumination in
the sample plane.These slits are placed after two
cylindrical lenses in each emission path. High numerical
aperture anisotropy imaging is feasible due to the
confocal arrangement. Acousto-optic tunable filter
(AOTF) is used to control laser power.
2. Primary laser sources are solid state lasers: emitting

488nm, 40nm5, 514nm; He-Ni lasers for 532nm and
633nm excitation wavelength respectively.
3. This set-up is also equipped with a separately steered

laser beam for patterned photo-bleaching.
4. Objectives: 63X (1.45NA), 20X (0.75NA), 10X (0.3NA),

20X (0.5NA), 40X (1.45NA).
5. Two linear array CCDs (dimension is 512 by 1) are used

to detect emitted fluorescence.
6. Appropriate emission filters were mounted in the

emission filter wheels in front of the linear array CCD
detectors.
7. For anisotropy measurements, a nanowire-based

polarization beam splitter (ProFlux™ polarizing
beamsplitter, Moxtek Inc., USA) is used mounted on the
main dichroic filter wheel in the emission path.
47
8. Extinction coefficient is 0.96 and G-Factor 0.68 (at
equivalent camera gains).
3.D: Hetero-FRET measurements
3.D.i: Theory
Förster Resonance Energy Transfer (FRET) is description of a

process where energy transfer from an excited donor (D) molecule to
an acceptor (A) happens through long range dipole-dipole coupling and
in a non-radiative fashion (Lakowicz, 1999). FRET can occur if certain
criteria are met:
1. Spectral overlap between donor emission spectrum and

acceptor absorption spectrum.
2. Quantum yield of the donor.
3. Distance between donor and acceptor molecule.

Molecular contact is not required for FRET to occur but
efficiency decreases sharply with respect to the distance,
as described below.
4. Geometric orientation between donor ad acceptor

transition dipoles.
Rate of energy transfer from D to A ( kT ) as derived from Förster theory
is given by following expression:
48
6
1  R0 
kT =
τ D  r 
or
Φ Dκ 2  9000(ln10) 
kT = J (λ )
τ D r 6  128π 5 Nn 4 
where, τ D = lifetime of donor
r = distance between donor and acceptor

R0 = Förster’s distance (at which efficiency of energy
transfer is 50%)
Φ D =Qantum yield of donor in absence of acceptor
κ 2 = orientaton factor depending on relative spatial

orientation of the donor emission dipole and acceptor
absorption dipole
N = Avogadro’s number
n = refraction index of the neighboring medium
J (λ ) = Spectral overlap integral that is measure of
overlap between normalized donor emission and
acceptor absorption spectrum
Spectral overlap integral is defined as:
∫F D (λ )ε A (λ )λ 4 d λ
J (λ ) = 0
∞
∫F
0
D (λ ) d λ
where, FD = Fluorescence intensity of donor
ε A = Molar extinction coefficient of the acceptor
49
λ = wavelength

R0 can be deduced as where R0 is in Α and J (λ ) is in M-1 cm3:
1
9.78 10 κ 2 n −4 Φ D J (λ )  6
R0 =× 3
The efficiency of energy transfer ( E ) is defined as the ratio of energy

transfer rate to the total decay rate of the donor:
kT
E=
τ + kT −1
D
where, τ D−1 = donor lifetime without acceptor, kT = rate of energy

transfer
Substituting kT in the above equation,
R06
E= 6 6
R0 + r
3.D.ii: Calculation of R0
1. Florescence emission spectrum were measured from

fluorescence spectrophotometer and normalized to its own
maxima ( FD (λ ) ).
2. Absorption spectrum of acceptor was analyzed to get molar

absorption coefficient for each wavelength ( ε A (λ ) ).
3. Spectral overlap was calculated from the equation below:
50
∞
∫F D (λ )ε A (λ )λ 4 d λ
J (λ ) = 0
∞
∫F 0
D (λ ) d λ
4. The R0 was then calculated for pairs of fluorophores (hetero-
FRET) or a single fluorophore (homo-FRET) using the

formula below.
1
9.78 10 κ n Φ D J (λ ) 
R0 =× 3 2 −4 6
3.D.iii: Measurement of FRET efficiency
The efficiency of Energy transfer ( E ) can be calculated by three

different ways:
1. Measuring donor fluorescence lifetime in presence and
absence of acceptor. Due to non-radiative energy transfer
donor fluorescence lifetime or quantum yield reduces
depending on the net amount of energy transfer being
transferred to acceptor. By this experimental technique,
one can obtain true efficiency of FRET.
2. Sensitized fluorescence emission: emission of acceptor

fluorescence in presence and absence of donor.
3. Varying acceptor to donor ratio: Keeping donor

fluorophore constant, donor fluorescence emission
changes in presence of varying acceptor concentration.
FRET efficiency can be calculated directly using following equations:
51
τ DA Φ F
E=
1− =
1 − DA =
1 − DA
τD ΦD FD
where, τ D = lifetime of donor in absence of acceptor
τ DA = lifetime of donor in presence of acceptor

Φ D =donor quantum yield in absence of acceptor
Φ DA =donor quantum yield in presence of acceptor
FD = fluorescence intensity of donor in absence of acceptor
FDA = fluorescence intensity of donor in presence of acceptor

3.D.iv: Hetero FRET measurements by donor lifetime
1. Lifetime of donor only (PLF) labeled FR expressing cells was

measured. PLF labeling was performed such as intensity
matches with respect when cells were labeled with both
donor and acceptor.
2. Photons were selected with band pass filter 500nm to 550nm

placed before MCP detector.
3. Lifetime parameters were obtained by fitting decay data as

described previously.
4. The efficiency of energy transfer is estimated by following

equation
 τ DA 
E % =−
1  ×100
 τ D 
where, τ D = average lifetime of donor in absence of acceptor
52
τ DA = average lifetime of donor in presence of acceptor

53
3.D.v: Varying donor to acceptor ratio – Hetero FRET

efficiency measurement
Since donor lifetime reduces as a result of FRET in presence of

acceptor, varying concentration of acceptor will result varying degree of
energy transfer that will be reflected in donor fluorescence lifetime.
1. Saturating concentration of fluorescent probe was prepared

by mixing two fluorophore (donor and acceptor) conjugated
ligand with varying ratio. Starting from saturated
concentration of donor only (D:A :: 1:0) to a ratio (D:A :: 1:X)
so that the donor signal was three fold higher than the
background count rate in the TCSPC measurement.
2. To obtain donor lifetime at the same donor intensity,

saturated donor labeling was titrated with unlabeled folic
acid. Donor only lifetime was measured at every point
matched with the intensity of donor with varying acceptor
labeling.
54
3.E: Reference
Becker, W. 2005. Advanced Time-Correlated Single Photon Counting

Techniques. Springer.
Grinvald, A., and I.Z. Steinberg. 1974. On the analysis of fluorescence
decay kinetics by the method of least-squares. Anal Biochem.
59:583-98.
Gryczynski, I., R.F. Steiner, and J.R. Lakowicz. 1991. Intensity and
anisotropy decays of the tyrosine calmodulin proteolytic
fragments, as studied by GHz frequency-domain fluorescence.
Biophys Chem. 39:69-78.
Krishna, M.M., A. Srivastava, and N. Periasamy. 2001. Rotational

dynamics of surface probes in lipid vesicles. Biophys Chem.
90:123-33.
Krishnamoorthy, G., N. Periasamy, and B. Venkataraman. 1987. On
the origin of heterogeneity of fluorescence decay kinetics of
reduced nicotinamide adenine dinucleotide. Biochem Biophys
Res Commun. 144:387-92.
Lakowicz, J. 1999. Principles of fluorescence spectroscopy. 2nd
Edition.
Lakshmikanth, G.S., and G. Krishnamoorthy. 1999. Solvent-exposed
tryptophans probe the dynamics at protein surfaces. Biophys J.
77:1100-6.
Rao, M., and S. Mayor. 2005. Use of Forster's resonance energy
transfer microscopy to study lipid rafts. Biochim Biophys Acta.
1746:221-33.
55
Swaminathan, R., U. Nath, J.B. Udgaonkar, N. Periasamy, and G.
Krishnamoorthy. 1996. Motional dynamics of a buried
tryptophan reveals the presence of partially structured forms
during denaturation of barstar. Biochemistry. 35:9150-7.
Varma, R., and S. Mayor. 1998. GPI-anchored proteins are organized
in submicron domains at the cell surface. Nature. 394:798-801.
Volkmer, A., V. Subramaniam, D.J. Birch, and T.M. Jovin. 2000. One-
and two-photon excited fluorescence lifetimes and anisotropy
decays of green fluorescent proteins. Biophys J. 78:1589-98.
Zipfel, W.R., R.M. Williams, and W.W. Webb. 2003. Nonlinear magic:
multiphoton microscopy in the biosciences. Nat Biotechnol.
21:1369-77.
56
Figure 3.A Microscope set up for FIAT and time-resolved fluorescence
measurements
Zeiss LSM 510 Meta microscope (Zeiss, Germany) equipped

with to steer a femtosecond 80.09 MHz (12 ns) pulsed Tsunami
Titanium:Sapphire (Ti:S) tunable multi-photon excitation laser
(Newport, Mountain View, CA). The Ti:S. The laser can be parked at a
single point for continuous illumination at a single point or scanned
across the field for collecting images. Time correlated single photon
counting (TCSPC) was accomplished using a Becker & Hickl 830 card
(Becker and Hickl, Berlin, Germany) as described (Becker, 2005).
Parallel ( I  ) and perpendicular ( I ⊥ ) emissions were collected
simultaneously into two Hamamatsu R3809U multi-channel plate

photomultiplier tubes (PMTs) using a polarizing beam splitter (Melles
Griot, Carlsbad, CA) to separate the parallel and perpendicular
components of the fluorescence emission, at the non de-scanned side.
This microscope with multiphoton excitation at non de-scanned
emission side has extinction ratio of 96% in the parallel beam path was
characteristic of the system.
57
Figure 3.B Illumination profile for single photon and multiphoton
fluorescence process
i. Example of single photon excitation of fluorescein solution by

focusing 488nm light with 0.16 NA objective. ii. Two-photon excitation
using 960nm wavelength pulsed femtosecond laser focused by 0.16
NA objective. (images taken from Nonlinear magic: multiphoton
microscopy in the biosciences. Nat Biotechnol. 21:1369-77.)
58
Figure 3.C Confirmation of two photon excitation
Fluorescein solution was taken on cover slip. It was excited at

790nm wavelength with varying laser power (incident peak photon flux
density). Laser power was measured at the back-focal plane of the
microscope. Concentration of the solution remains constant during the
time period of the measurement. Graph shows logarithmic plot of
fluorescence emission intensity versus excitation power, obeyed a
power-squared intensity dependence as indicated by the measured
slope of 1.98, thereby confirming the existence of the two-photon
excitation
59
Figure 3.D Initial anisotropy values for different mode of excitation
i. Cone of excitation for one, two, and three photon process. ii.
Excited state distribution for r0 one, two, and three photon excitation.
(taken from: http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html)
60
Figure 3.E Principle of time correlated single photon counting (TCSPC)
The sample is excited with a pulse of light (the red line),

resulting in the fluorescence photon (green graph) waveform shown at
the top of the figure. This is the wave-form that would be observed
when many fluorophores are excited and numerous photons are
observed over multiple such events. TCSPC the conditions are
adjusted so that less than one photon is detected per laser pulse. In
fact, the detection rate is typically 1 photon per 100 excitation pulses.
The time is measured between the excitation pulse and the observed
photon and stored in a histogram. The x-axis is the time difference and
the y-axis the number of photons detected for this time difference.
61
Figure 3.F Instrument response function (IRF)
Graph shows instrument response function obtained from the

TCSPC setup. Instantaneous scattered photons from 20-40nm gold
particles on a cover slip were directed to MCP-PMTs to measure the
IRF. The FWHM is 60ps for this system.
62
Figure 3.G Multiphoton illumination profile
200nm beads imaged with single photon confocal (i, green) and
multiphoton (ii, red) is overlapping with each other (iii, yellow). Uniform
fluorescence intensity profile obtained when fluorescein solution was
imaged with multiphoton excitation (iv). Both images confirm that the
multiphoton excitation and collection light path are aligned with respect
to the system for imaging purpose. Both criteria are important for
consistent anisotropy measurements. Scale bar is 10µm.
63
Figure 3.H Examples of time- resolved fluorescence decays
i. Fuorescence intensity decays for fluorescein dye at pH11

fitted with one exponent decay using iterative re-convolution fitting
routine. This fitting shows 4.01±0.2ns lifetime of fluorescein at ph11. ii.
Fluorescence anisotropy decay of fluorescein dye in water at pH11.
Rotational correlation time was obtained by fitting with one exponential
decay using iterative re-convolution routine and the value is 120±10ps.
64
Figure 3.I Line-scanning confocal LSM 5 Live microscope
Custom-designed line-scanning LSM 5 Live microscope (Zeiss,

Jena, Germany) adapted for fluorescence polarization measurements.
This set-up is also equipped with a separately steered laser beam for
patterned photo-bleaching. For the purpose of anisotropy
measurements, main dichroic in the emission path was replaced with a
nanowire-based polarization beam splitter (ProFlux™ polarizing
beamsplitter, Moxtek Inc., USA), and matched emission filters were
mounted in the emission filter wheels in front of the linear array CCD
detectors. The spatial resolution achievable here is 230nm in x-y and
660nm in z (using 1.4NA, 63X objective for 495-530nm fluorescence
emission), and high numerical aperture anisotropy imaging is feasible

due to the confocal arrangement.
65
Chapter 4
Aerolysin toxin alters GPI-Anchored Protein

organization at the cell surface
4.A: Introduction
Many pathogenic organisms produce toxic substances which

are able to disrupt cellular membranes causing perturbations in the
host system. The plasma membrane of a cell acts as a barrier to
protect from such substances. But plasma membrane is complex

system constituting lipids and proteins. As evident from recent studies
(see preceding Chapters 5 and 6) that the organization of plasma
membrane components is necessary to accomplish multiple functions.
Bacterial pathogens have also used this form of organization at the
plasma membrane as targets and eventually cause its perturbation
(Lafont et al., 2004).
Various bacteria produce different types of toxins. Amongst

them, the largest toxin class (30% of all types) is pore-forming toxins
(PFTs) (Joseph E. Alouf, 2006). PFTs are also produced by higher
organisms. These toxins are secreted as soluble proteins and they
form a transmembrane channel in the target cell membrane. Firstly, the
soluble toxin molecules come toward cells that express a target
receptor on the membrane. Receptors bound PFTs then oligomerize,
followed by membrane insertion leading to channel formation in the
membrane (Parker and Feil, 2005). There are two ways toxin can cross
the membrane as classified alpha helical (α-PFT) or β-barrel (β-PFT).
α-PFTs are predicted to be helical and span lipid bilayers. It contains
66
Aerolysin and GPI-APs
stretches of hydrophobicity. In contrast, β-PFTs are not predicted to be
transmembrane based on the analysis of hydrophobicity. They
construct pairs of amphipathic β-strands. Upon oligomerization (multi-
protein structures), membrane insertion happens by creating a
hydrophobic surface.
The toxin of interest in my studies is produced by Aeromonas

hyhrophila , and is called Aerolysin. This toxin belongs to the second
category that is β -PFT. A. hydrophila secretes a precursor toxin,
proaerolysin, via a type II secretion system in the extra-cellular medium
(Buckley, 1990). Proaerolysin binds to the glycosyl phosphatidyl inositol
(GPI)-anchored proteins on membrane (Cowell et al., 1997; Fivaz et
al., 1999; Fivaz et al., 2002; Hong et al., 2002) . The binding property
depends on the glycan core and the N-linked sugar moiety of the
receptors (Abrami et al., 2002; Hong et al., 2002). Proaerolysin is
activated to aerolysin by removing 40 amino-acids from the C-terminus
(van der Goot et al., 1992). This is done by gut proteases, Aeromonas
proteases or members of the furin family of mammalian endoproteases
(Abrami et al., 1998) in nature. Aerolysin will subsequently oligomerize
into a heptameric ring (Wilmsen et al., 1992); if the concentration is
high, it can oligomerize without binding to its receptor.
The X-ray structure of proaerolysin in the dimeric form revealed

an L-shaped elongated molecule divided into two domains, a small
globular domain, and a large lobe linked together by a long stretch of
residues (Parker 1994). The globular domain, domain 1, is responsible
for binding the toxin to sugar modifications of the receptor (Hong et al.,
2002). The elongated domain of the protein is sub-divided into three
distinct domains. Domain 2 is involved in binding to the GPI-anchor
(MacKenzie et al., 1999) which eventually initiates the oligomerization
process (MacKenzie et al., 1999). Heptameric state is achieved
67
through interaction between domain 3 of each monomer too some
extent (Lesieur et al., 1999). There is a loop structure which actually
helps the part of the protein that get inserted into the membrane to
form the transmembrane amphipathic β-barrel (Iacovache et al., 2006;
Melton et al., 2004). The last part, domain 4 contains the pro-peptide,
which is proteolytically removed upon activation (van der Goot et al.,
1994). No high-resolution structure is available for aerolysin in the
transmembrane state. However, cryo-negative staining EM analysis
has performed on the heptameric form of an aerolysin mutant (Tsitrin et
al., 2002); a single point mutation Y221G which converts the wild-type
aerolysin hydrophobic heptamer into a water-soluble complex. Studies
show a conserved mushroom-shaped soluble heptamer (Figure 1.A),
that matches the structure obtained by 2D crystallography determined

from pores of wild type aerolysin (Parker and Feil, 2005; Wilmsen et
al., 1992).
GPI-anchored proteins are present in cholesterol sensitive

nanometer scale clusters of size 2-4 molecules at the plasma
membrane in living cells. After activation by carboxy-terminal cleavage,
soluble aerolysin binds GPI-anchored proteins in the membrane to
form heptameric pore-forming complexes. Thus upon binding, aerolysin
must alters the organization of GPI-anchored proteins at the cell
surface. Here, I attempt to investigate properties of this altered
organization. I use Föster’s resonance energy transfer (FRET) to
extract nanometer length-scale structural information about optically
unresolved aerolysin induced GPI-anchored proteins complexes in
CHO cells expressing a model GPI-anchored protein, GFP-GPI or the
folate receptor (FR). Using steady state and time resolved
measurement to determine the extent of homo- and hetero-FRET, I find
that the relative fraction and size of aerolysin bound GPI-anchored
protein clusters are larger than pre-existing GPI-anchored protein
68
clusters. I have also compared the theoretically-modeled hetero-FRET
efficiency with the experimentally observed hetero-FRET efficiencies to
obtain a size of the aerolysin induced structures. Results indicate that
aerolysin reorganizes GPI-anchored proteins to form higher-order
oligomers. These structures could have consequences for the function
of GPI-anchored protein in terms of altered trafficking and signaling
behavior.
4.B: Results
I have used wide-field epifluorescence microscope and a

custom designed multiphoton based laser scanning microscope

(described in Chapter 3.C.i) for anisotropy imaging of emitted
fluorescence from cells. Cells, expressing FR were labeled with PLF
(Nα-pteroyl-Nε-(4’-fluoresceinthiocarbamoyl)-L-lysine and / or PLR (Nε-
pteroyl-Nα-(4’-lissamine rhodamine thiocarbamoyl)-L-lysine), a
fluorophore tagged ligand to FR and expressing GFP-GPI were used
for experiments.
4.B.i: Uniform Surface Distribution of GPI-APs
Both low and high resolution (where lateral and axial resolution
being 260nm and 890nm respectively) fluorescence intensity image
showed that uniform distribution of GPI-anchored protein on the cell
surface. Cells expressing GFP-GPI or FR-GPI (surface labeled with
various fluorescent analogue of folic acid) were imaged in its
corresponding fluorescent emission wavelength range. It is evident
from the fluorescent images in Figure 4.B.i (widefield) and Figure 4.B.ii
(multiphoton confocal) that upon treatment with activated (nicked)
aerolysin toxin mutant (Y221G; (Fivaz et al., 2002)) between 0.5 to
69
1µg/ml, the optically resolvable surface distribution of GPI-anchored
protein at light microscopic resolution remain unaltered.
4.B.ii: Aerolysin induces alteration in GPI-AP organization on

cell surface
Using homo-FRET method, previously, it was shown that GPI-

anchored proteins form nanoclusters at the cell surface (Sharma et al.,
2004). I have done homo-FRET measurements on cells treated with
aerolysin. Both wide-field and confocal steady state anisotropy
measurement showed depolarized value compared to control cells
(Figure 4.C.i - wide-field; Figure 4.C.ii- multiphoton confocal). Wide-

field 20X imaging of PLF-labeled IA2.2 cells showed 0.02 unit decrease
in anisotropy value in aerolysin bound cell surface with respect to the
control cells. In case of multiphoton excited GFP-GPI expressing cells
the fluorescence emission was additionally depolarized; aerolysin
bound cell surface exhibited a value of fluorescence anisotropy that
was 0.24±0.005 with respect to the control cells, 0.26±0.004, with a
63X 1.4NA objective. This data implies that aerolysin alters the
organization of GPI-anchored protein on cell surface.
A further depolarized anisotropy value could indicate either

presence of larger and compact clusters or higher fraction of clustered
population of GPI-anchored proteins, as a result of aerolysin induced
clustering process. However, steady state anisotropy measurement
cannot provide a resolution of these possibilities; additional
depolarization caused due to any changes in the rotational properties
after aerolysin binding could also alter any simple extrapolation
between anisotropy values in the bond and unbound states.
70
4.B.iii: Aerolysin induced GPI-AP clusters are compact
Time resolved anisotropy measurements on GFP-GPI

expressing cells confirmed that the enhanced depolarization was due
to increaased homo-FRET. The fast rotational decay rate, a signature
of the FRET process was enhanced by 39%. Its amplitude reflects the
fraction of clusters present, and there is a corresponding increase in
this amplitude by 75% compared to the control cells (Figure 4.D.i;
Table 4.A). This data suggests that aerolysin induced GPI-AP clusters
are more compact and involves larger fraction of monomers in a
confocal volume. Furthermore the second rotational correlation time
remain similar suggesting additional depolarization is due to FRET and

not due any alteration in molecular rotation of the GFP monomer.
4.B.iv: Confirmation of higher order organization with

fluorescence lifetime measurements
CHO cells, stably expressing folate receptors, were labeled with

PLF –donor and/or PLR –acceptor. Cells were treated with 1.5µg/ml
activated (nicked) aerolysin on ice for 1hr. before fluorescent lifetime
measurements. Fluorescence lifetime decays were obtained from
donor photons from whole cell with high time resolution. This high
precision measurement showed aerolysin treated cells have reduced
donor lifetime compared to control cell in presence of acceptor (Table
4.B; Figure 4.E).
71
4.B.v: Estimation of cluster size and fraction can be done by

theoretical modeling of hetero-FRET
Efficiency of hetero-FRET can be estimated by donor lifetime in

presence and absence of acceptor; FRET causes a measurable
decrease of the donor lifetime. Theoretical prediction (explained in next
section) of efficiency of energy transfer versus increasing acceptor to
donor ratios can be used to determine cluster characteristics (Sharma
et al., 2004) (Figure 4.F). Cells expressing FR were taken and treated
with aerolysin. Saturating concentration of fluorescent probe was
prepared by mixing two fluorophore (donor and acceptor) conjugated
ligand with varying ratio. Starting from saturated concentration of donor

only (PLF:PLA :: 1:0) to a ratio (PLF:PLR:: 1:0.5). Both aerolysin
treated and untreated cells were labeled with varying ratio (PLF:PLR)
of the probe mixture. To compare actual labeling ratios for donor and
acceptor, images were taken in both channels for PLF and PLR. Since
lifetime measurement may vary when signal to noise ratio decreases,
PLF supplemented with varying concentration of unlabelled folic acid
was also used to measure donor lifetime in each case having
approximately same intensity as compared to PLF-PLR labeled cells.
Lifetime was measured from 1µm2 area on the cell surface. Hetero-
FRET efficiency of was calculated in for each labeling ratios. Acceptor
to donor ratio versus efficiency plot was obtained. The data show that
aerolysin induced clusters display a steeper rise in energy transfer
efficiency consistent with the greater depolarization rate due to homo-
FRET, compared to pre-existing GPI-anchored protein nanoclusters
(Figure 4.F). Thus, aerolysin induced clusters are compact, contain
more number of monomers and larger fraction of molecules are in
clustered form compared to native clusters.
72
Explaining the theoretical calculation for determining hetero-FRET
efficiency variation with cluster size and fraction:
According to previously published work from our laboratory (Sharma et

al., 2004), done by Dr. Sarasij RC, in an experiment let us consider a
cluster containing n proteins labeled as m acceptors (A) and (n − m)
donors (D) separated by distances of order R0 (determined for a
specific D-A pair). In this experiment, the n and m vary gradually. For
instance, if the probability of having an A or D in a given cluster is
equal, then the relative abundance amongst all clusters of size n with
at least one donor is
=Pnm n
Cm / (2n − 1)
n!
where n
Cm = is the combination of m acceptors from a
n !( n − m ) !
cluster of size n . For example, when n =3 , the probability of

occurrence of DDD, ADD and AAD are 1/7, 3/7, 3/7 respectively).
The excited donor can transfer its energy to another donor with
probability p : D∗ → D (homo-FRET), to an acceptor with
probability q : D∗ → A or emitted as fluorescence with probability r . We

know that p + q + r =
1 . The possibility that an excited acceptor returns
its energy to a donor ( s : A∗ → D ) is ignored (s = 0). The quantities p, q

can be obtained from the overlap of the respective emission and
absorption spectra of the fluorophores, D and A, and then r can be
estimated. Obviously, it will be varying with different set of
fluorophores.
The observed emission from A∗ following a transfer from D∗ → A . If

Wn is the probability that the excitation of a donor D∗ in a cluster of
73
size n is never transferred to A , then the likelihood of observing the
hetero-FRET signal is
Z = x2 (1 − W2 ) + x3 (1 − W3 ) + x4 (1 − W4 ) + ...
Now, for very small clusters there is less probability that donor
finds an acceptor to transfer its energy contributing to hetero-FRET
compared to finding another donor and do homo-FRET. In this
scenario, if we change the strength of probability of finding an acceptor
by increasing the acceptor to donor ratio, then, when donor to acceptor
ratio is d , the relative abundance of cluster of size n consisting m
acceptors and n − m donor molecules. The probability becomes
n
Cm d n − m (1 − d ) m
Pnm =
1 − (1 − d ) n
To calculate Wn split it into (n − 1) parts,
Wn = Wn0 + Wn1 + Wn2 + ... + Wnn −1
Where the superscript is the number of acceptor in the cluster. If

m = 0 then donor cannot transfer its energy to the acceptor an hence
Wn0 = Pn0
And if m= n − 1 , then a) the donor can emit its energy as

fluorescence unless hetero-FRET has occurred; b) if the donor has not
transferred its energy to any of the acceptors, then n − 1 independent
events that it does not transfer its energy to each of the n − 1 acceptor
in the cluster. So taken together,
n −1
=
Wn Pnn −1 (1 − q ) n −1
74
For other values of m ,
 r 
Wnm = Pnm  
 1−α 
α =(1 − r )(1 − q) m
This how each solid lines is generated for expected energy

transfer efficiencies for different donor to acceptor ratios where cluster
size and abundance of clusters are varied in Figure 4.F.
75
4.C: Discussion
There is significant re-organization of GPI-APs observed after binding

of aerolysin molecules to the GPI-anchor on living cell surface.
Further depolarization in anisotropy implies more molecules in close

proximity. It could either be increase in percentage of closely packed
structures or number of molecules involved per induced cluster or both.
Time resolved anisotropy measurements show that there is more close

packing of molecules, as well as the fraction of molecules undergoing
FRET is increased after treatment.
Hetero-FRET data confirms Homo-FRET-based results about re-

organization of GPI-APs. Predicted theoretical model for hetero-FRET
can be used further for extracting the information about the cluster
characteristics.
76
4.D: References
Abrami, L., M. Fivaz, E. Decroly, N.G. Seidah, F. Jean, G. Thomas,

S.H. Leppla, J.T. Buckley, and F.G. van der Goot. 1998. The
pore-forming toxin proaerolysin is activated by furin. J Biol
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Abrami, L., M.C. Velluz, Y. Hong, K. Ohishi, A. Mehlert, M. Ferguson,
T. Kinoshita, and F. Gisou van der Goot. 2002. The glycan core
of GPI-anchored proteins modulates aerolysin binding but is not
sufficient: the polypeptide moiety is required for the toxin-
receptor interaction. FEBS Lett. 512:249-54.
Buckley, J.T. 1990. Purification of cloned proaerolysin released by a
low protease mutant of Aeromonas salmonicida. Biochem Cell

Biol. 68:221-4.
Cowell, S., W. Aschauer, H.J. Gruber, K.L. Nelson, and J.T. Buckley.
1997. The erythrocyte receptor for the channel-forming toxin
aerolysin is a novel glycosylphosphatidylinositol-anchored
protein. Mol Microbiol. 25:343-50.
Fivaz, M., M.C. Velluz, and F.G. van der Goot. 1999. Dimer
dissociation of the pore-forming toxin aerolysin precedes
receptor binding. J Biol Chem. 274:37705-8.
Fivaz, M., F. Vilbois, S. Thurnheer, C. Pasquali, L. Abrami, P.E. Bickel,
R.G. Parton, and F.G. van der Goot. 2002. Differential sorting
and fate of endocytosed GPI-anchored proteins. Embo J.
21:3989-4000.
Hong, Y., K. Ohishi, N. Inoue, J.Y. Kang, H. Shime, Y. Horiguchi, F.G.
van der Goot, N. Sugimoto, and T. Kinoshita. 2002.
Requirement of N-glycan on GPI-anchored proteins for efficient
binding of aerolysin but not Clostridium septicum alpha-toxin.
Embo J. 21:5047-56.
77
Iacovache, I., P. Paumard, H. Scheib, C. Lesieur, N. Sakai, S. Matile,
M.W. Parker, and F.G. van der Goot. 2006. A rivet model for
channel formation by aerolysin-like pore-forming toxins. Embo J.
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Joseph E. Alouf, M.R.P. 2006. The Comprehensive Sourcebook of
Bacterial Protein Toxins. Academic Press.
Lafont, F., L. Abrami, and F.G. van der Goot. 2004. Bacterial
subversion of lipid rafts. Curr Opin Microbiol. 7:4-10.
Lesieur, C., S. Frutiger, G. Hughes, R. Kellner, F. Pattus, and F.G. van
der Goot. 1999. Increased stability upon heptamerization of the
pore-forming toxin aerolysin. J Biol Chem. 274:36722-8.
MacKenzie, C.R., T. Hirama, and J.T. Buckley. 1999. Analysis of
receptor binding by the channel-forming toxin aerolysin using

surface plasmon resonance. J Biol Chem. 274:22604-9.
Melton, J.A., M.W. Parker, J. Rossjohn, J.T. Buckley, and R.K. Tweten.
2004. The identification and structure of the membrane-
spanning domain of the Clostridium septicum alpha toxin. J Biol
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Parker, M.W., and S.C. Feil. 2005. Pore-forming protein toxins: from
structure to function. Prog Biophys Mol Biol. 88:91-142.
Tsitrin, Y., C.J. Morton, C. el-Bez, P. Paumard, M.C. Velluz, M. Adrian,
J. Dubochet, M.W. Parker, S. Lanzavecchia, and F.G. van der
Goot. 2002. Conversion of a transmembrane to a water-soluble
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van der Goot, F.G., K.R. Hardie, M.W. Parker, and J.T. Buckley. 1994.
The C-terminal peptide produced upon proteolytic activation of
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the cytolytic toxin aerolysin is not involved in channel formation.
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van der Goot, F.G., J. Lakey, F. Pattus, C.M. Kay, O. Sorokine, A. Van
Dorsselaer, and J.T. Buckley. 1992. Spectroscopic study of the
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79
Table 4.A: TRA data for homo-FRET measurement
n τ r1 τr2 τ F1 τF2
χ 2
r0 rss
( ar1 ) ( ar 2 ) ( aF 1 ) ( aF 2 )
GG8
0.22 ± 2.7 ±
Control 0.38 34.5 ± 2
1.1 ± 0.44 ± 0.02 0.05 0.9 ± 0.1
Cell 6 ± (30.92 ±
0.057 0.005 (30.08 ± (0.7 ± (0.3±0.04)
mean ± 0.007 0.01)
0.013) 0.04)
s.d.
Aerolysin
0.14 ± 29.8 ± 2.8 ±
treated 0.36 0.9 ± 0.09
1.2 ± 0.44 ± 0.032 2.6 0.11
GG8 cells 7 ± (0.36 ±
0.07 0.006 (0.12 ± (0.88 ± (0.64 ±
mean ± 0.010 0.041)
0.02) 0.02) 0.041)

s.d.
n : number of sample; χ 2 : chi-square value from the fit; r0 : intial
anisotropy; rss : steady state anisotropy obtained from the fit; τ r1 : fast
rotational time; ar1 : amplitude of the fast rotational component; τ r 2 : slow
rotational time; ar 2 : amplitude of the slow rotational component; τ F 1 :
fluorescence lifetime 1; aF 1 : amplitude of the lifetime 1; τ F 2 : fluorescence
lifetime 2; aF 2 : amplitude of the lifetime 2.
80
Table 4.B: Donor fluorescence lifetime with varying A/D
D A A/D Treatment n# τavg †† (ns)
1.95
PLF 0 10
(±0.03)
1.8
PLF PLA 0.5 31
(±0.03)
1.7
PLF PLA 1 11
(±0.09)
1.59
PLF PLA 2 28
(±0.09)
2.03
PLF 0 Aerolysin 16
(±0.09)
1.814
PLF PLA 0.5 Aerolysin 33
(±0.064)
1.6
PLF PLA 1 Aerolysin 11
(±0.027)
1.56
PLF PLA 2 Aerolysin 21
(±0.08)
††
Average fluorescence lifetime was calculated from the following
equation: τ av = ( A1 ×τ 1 ) + ( A2 ×τ 2 ) . Where, τ 1 : fluorescence lifetime 1; a1 :
amplitude of the lifetime 1; τ 2 : fluorescence lifetime 2; a2 : amplitude of
the lifetime 2 obtained as fit parameters.
81
Figure 4.A: Probable structure of aerolysin complex
i) A simple three-dimensional model for the complex. In this, the

strongly stain-excluding central ring becomes a cylinder projecting from
one side of the 'wheel-like' structure containing seven arms, each
having two globular domains. The cylinder is likely to contain the ion
channel and will presumably integrate into the membrane. The outer
arms, which may be flexible since they exhibit variable curvature when
seen in side view, do not penetrate the membrane but lie close to it.
ii) The fit of the aerolysin monomers into the channel density
obtained from the image of the aerolysin channel derived from the
0
electron microscopy. The resolution of the image is 25 Α . The image
consists of a central cylindrical-shaped density of outer diameter
0 0
~46 Α encircling a water-filled channel 17 Α in diameter.
82
Figure 4.B: Distribution of GPI-anchored protein on cell surface
Multiphoton confocal images of labeled folic acid receptor

expressed on cell surface treated or not treated with aerolysin.
Distribution of GPI-anchored folate receptor is consistent with a
random distribution of monomers or clusters that are smaller than the
diffraction limit of an optical microscope. No further change in
distribution of GPI-AP was observed after aerolysin treatment.
83
Figure 4.C: Steady-state anisotropy measurement on cell surface
CHO cells stably expressing GFP-GPI or FR-GPI were treated

with 1.5µg/ml activated (nicked) aerolysin on ice for 1hr. Steady state
anisotropy imaging was performed in wide field microscope (20X
objective) for cell expressing FR-GPI labeled with PLF (i). Same was
done using multiphoton confocal microscope (in TCSPC mode) for
GFP-expressing cells (ii). In both cases, intensity independent
anisotropy distribution was obtained.
84
Figure 4.D: Time resolved anisotropy decays for GPI-AP organization
Time resolved anisotropy experiment was performed on GFP-

GPI expressing cells as control and after aerolysin treatment. The
enhanced depolarization observed in steady-state measurements is
validated to be due to enhanced homo-FRET. The rate and the
amplitude of fast depolarization component (due to homo-FRET)
obtained from time resolved anisotropy decay is higher (decay rate is
increased by 39% and corresponding increase in amplitude by 75%)
than that of the control cells suggesting aerolysin induced GPI-AP
clusters are more compact and larger than the pre-existing
organization.
85
Figure 4.E: Hetero FRET observed by fluorescence lifetime
Fluorescence lifetime decays are obtained from donor

fluorescence photons from an area of 1µm2 at cell surface where FR-
GPI anchored proteins are labeled with PLF (donor) and PLA546
(acceptor) ratio 1:1. This high precision measurement shows decrease
in donor-lifetime in boh donor-acceptor labeled cells (green graph)
compared to donor alone (violet graph) labeled cells which estimates
detectable hetero-FRET in GPI-AP clusters at the cell surface. Upon
treatment with aerolysin, the donor lifetime decreases further (red
graph). In Aerolysin treated cells a 11% decrease in donor lifetime has
estimated compared to cells without any toxin-treatment.
86
Figure 4.F: Energy transfer efficiency changes with D to A ratio
Lines with various colors correspond to the theoretical

predictions with the clusters (20% and 40% molecules are in clusters)
composed entirely of dimmers or trimers or quadramers. Fuorescence
lifetime decay was obtained from a 1µm2 area on the cell surface by
scanning the ROI with multiphoton. Black and red points joined with
dotted lines show experimental results from control and aerolysin
treated cells respectively. Donor lifetime was calculated where
acceptor to donor (PLA and PLF respectively) ratio was varied from 0:1
to 2:1. The efficiency of hetero-FRET was calculated for each case.
This result shows that aerolysin induced clusters display different
nature of graph compared to existing theoretical predictions and control

experimental graph. This data proves that aerolysin induced clusters of
GPI-anchored proteins are bigger than that of the clusters present on
untreated cells.
87
Chapter 5
GPI-anchored protein nano-clusters are immobile

and heterogeneously distributed on living cell
membranes
5.A: Introduction
Since the last decade, many interesting aspects have been

brought up in the field of protein and lipid diffusion at the cell
membrane. Various research groups, using different techniques, have
proposed different models for plasma membrane diffusion (Bacia and

Schwille, 2007; Chen et al., 2006; Kenworthy, 2007; Lommerse et al.,
2004; Mayor and Rao, 2004; Rao and Mayor, 2005). The hop-diffusion
model proposed by Akihiro Kusumi (Fujiwara et al., 2002) has provided
compelling evidence for hop diffusion where membrane components
experience the confining aspect of an underlying cytoskeleton. But
conflicting evidence from other laboratories regarding the underlying
basis for hop diffusion leaves scope of debate. All these findings,
however, critically depend on the spatio-temporal scale of
measurements. Since it has been established GPI-anchored proteins,
are found in cholesterol dependent sub-resolution clusters at the cell
surface (Sharma et al., 2004; Varma and Mayor, 1998), several studies
have attempted to explore the mobility and organization of these
molecules in an attempt to understand the properties of GPI-anchored
proteins.
Since, the identification of sub-resolution clusters is not direct, it

is very difficult to study its dynamics. It is also important to realize that
cluster mobility, molecular association kinetics and its dynamics are
88
Nanocluster dynamics I
inter-related. In this context, it is important to use the correct technique
and procedure to measure the properties of the clusters at the cell
surface.
In our laboratory, it was shown that GPI-anchored proteins are

distributed as monomers and sub-resolution clusters on the surface of
living cells (Varma and Mayor, 1998). This information was obtained
using wide-field steady-state low-resolution anisotropy imaging on cells
expressing both FR-GPI and GFP-GPI. The fluorescence anisotropy
property of a randomly distributed sample should ideally have density
dependent profile, as shown in case of Rhodamine 6G (Figure 2.D.i),
where the average inter-fluorophore distance decreases (one criterion
for FRET to occur) as concentration of fluorophore increases.

Surprisingly, the anisotropy profile obtained from cells, containing a 40-
fold range of protein levels on the cell surface, was independent of
protein concentration (as measured from the range of fluorescence
intensity) and lower than the value obtained for protein in isolation.
These observations confirm the existence of sub-resolution clusters
that potentially violate the law of mass action. Using both time resolve
anisotropy decay data and a model explaining the bleaching-profile of
fluorophores attached to the receptors, the characteristic of GPI-AP
organization at the cell surface was elucidated (Sharma et al., 2004).
This theoretical model suggests that at the cell surface, 20-40% of the
proteins are present as nanocluster which on an average consists of 2-
4 molecules (Sharma et al., 2004). Furthermore, high resolution wide-
field anisotropy imaging (Goswami et al., 2008) reveals that nano-
clusters are distributed heterogeneously on the cell surface.
These results provided an average picture of the cel surface, but

poorly resolved the spatial distribution of GPI-AP organization, and
clusters to monomer ratio, and gave no information regarding the
89
distribution and exchange between monomer and clusters. To
understand further details about the distribution and maintenance of
clusters at the cell surface, it was necessary to use anisotropy imaging
with high spatial and temporal resolution. A custom designed line-
scanning confocal microscope capable of capturing high-resolution
time-lapse anisotropy images (described in Chapter 3) of emission
fluorescence from cells is used for this purpose. The existence of
heterogeneous, non-random distribution of GPI-AP clusters on baso-
lateral membrane of cell is observed and validated by statistical
distribution analysis. A new assay, Anisotropy Recovery After
Photobleaching (ARAP) is developed using the same microscope to
understand the mobility of GPI-AP clusters and monomers at the cell
surface.
90
5.B: Results
I used a custom designed line-scanning microscope (described

in Chapter 3) with high spatio-temporal resolution for time-lapse
anisotropy measurements of emission fluorescence.
Cells, expressing FR were labeled (methods and materials

Chapter 2) with PLBTMR (Nα-pteroyl-Nε-(4’-BodipyTMR)-L-lysine - a more
stable fluorophore with respect to PLF), a fluorophore tagged ligand to
FR for experiment. PLBTMR has already been characterized on cells in
terms of its ability to bind specifically to the membrane bound receptor
and the pathway of internalization (work done by Subhasri Ghosh in

the laboratory).
5.B.i: GPI-AP clusters are preferentially distributed in certain

regions of the cell surface
High resolution (where lateral and axial resolution being 260nm

and 890nm respectively) anisotropy imaging was performed on cells
with PLBTMR-FR-GPI with 60X 1.4NA objective on a line scanning
microscope. Confocal anisotropy images have shown two distinct type
of optically resolvable regions on the cell surface – a. low average
fluorescence anisotropy regions and these are generally associated
with flat regions of the cell membrane, can be defined as flat cellscapes
[Figure 5.A; boxes (i-ii)]; b. regions with high average anisotropy,
associated with membrane overlying protrusive actin architecture such
as dynamic cell edges, such as membrane ruffles or leading edges of
lamellipodia [Figure 5.A; boxes (iii-iv)]. Such a description matches with
previous anisotropy map of cell in wide-field microscope (work done by
Sameera Bilgrami in the laboratory). Clearly, in case of confocal
anisotropy images, single plane of membrane reports higher
91
differences in average anisotropy in these two categories of region
compared to wide-field images, where range of difference in anisotropy
is being diluted out because of differential spatial distribution of
receptor organization on both apical and basolateral membrane. Such
distinct distribution is always present on the cells were kept in both
room and physiological temperature (37°C: Figure 5.A.i and 22°C:
5.A.vi).
5.B.ii: Non-random distribution of sub-resolution clusters of

GPI-AP on flat cellscapes
Statistical analysis was performed on the sub-resolution cluster

distribution by examining the anisotropy distribution at the level of

individual pixels (since anisotropy is a linear combination of clustered
and monomeric population) in the flat cellscapes from a number of cells
kept at 37 °C. It shows that there is a typical anisotropy distribution
(pixel wise) from flat part of cell membrane since multiple distributions
overlap with the normalised anisotropy distribution (Figure 5.B.i). To
determine whether the nanoclusters are spatially correlated or
randomly distributed, the observed distribution of anisotropy was
compared with the expected distribution by modelling the nanoclusters
and monomers according to a Poisson process.
The experimental values of mean anisotropy A and mean intensity
I yield a mean intensity of clusters Ic and mean intensity of
monomers I m . These are given by the following equations:
( A − Ac ) I
Im =
( Am − Ac )
( A − Am ) I
Ic =
( Ac − Am )
92
I m and I c are related to the number of monomers N m and
number of clusters N c via the intensity per fluorophore i
Ic = i Nc
Im = i Nm
If N molecules are scattered randomly on a lattice of L sites, the

probability of a site having n molecules is given by the Poisson
e− ( n ) n( n )
distribution, P (n) = , where n = N . It follows that Nc
n! L
and N m , and therefore I c and I m will independently follow similar
distributions.
We generate strings { I m } , { I c } of intensity values due to
monomers and clusters by drawing random numbers from a Poisson

distribution with means given by I m and I c . These strings are then
used to generate a string of anisotropy values using
Am { I m } + Ac { I c }
{ A} =
{I m } + {I c }
A large enough string provides a well averaged anisotropy

distribution, with which we compare the experimental data. Although,
the distribution analysis of nanoclusters are similar to that of obtained
previously from wide-field images (Goswami et al., 2008), confocal
images provide higher resolution and dynamic range in X-Y-Z planes
and in anisotropy values respectively. The long exponential tail (linear
decay slope in the log plot: shown by the black line in Figure 5.B.ii) is
distinct from both the generated Poissonian distribution (Figure 5.B.ii;
93
green line) , as well as solution of GFP (Figure 5.B.ii; blue dots). The
latter two distributions are typical of spatio-temporally uncorrelated
Gaussian random processes. The exponential tails indicates the
existence of correlations in the distributions of nanoclusters from a
single membrane. These results suggest that while nanoclusters are
present at the cell surface, they are not randomly distributed.
5.B.iii: Nanosclusters are immobile
To study the dynamics of the nanoclusters we utilized a

photobleaching-based perturbation strategy. Cell (PLBTMR-FR-GPI)
images were acquired at 20°C on a line scanning microscope with 60X,

1.4 NA objective. Initially, there is a significant depolarization of
fluorescence emission detected in the whole illuminated area (Figure
5.D; Pre-Bleach) which is a characteristic of the steady state
distribution of nanoclusters and monomers. A region 1μm2 was chosen
for bleaching as shown in the first panel of Figure 5.C and average
80% fluorophore was bleached within that area using high-intensity
pulse from a separate bleaching laser. Following the bleaching of
PLBTMR-FR-GPI at the centre of the illuminated area (Figure 5.C;
Bleach, magenta box), it is found that while the fluorescence intensity
recovers (Figure 5.C; Post-Bleach 1 and 4 min), the anisotropy in the
bleached spot does not (Fig.2C; Post-Bleach 1 and 4 min). Images
were taken after one and four minute post bleaching. Notice that in the
regions surrounding the bleached areas, the intensity and anisotropy
remain unchanged during this time (Figure 5.C; brown and blue boxes).
Quantitative analysis from multiple runs of the same experiment
(anisotropy recovery after photo-bleaching or ARAP) confirms this
observation (Figure 5.D). Increase in intensity and high anisotropy
values in the photo-bleached region post recovery may be explained
only if monomeric species diffuse in the bleached area from the
94
neighboring regions and that neither nanoclusters are mobile nor do
they reform at this temperature within 4 minute timescale at 20°C.
In contrast at 37°C, in a similar ARAP experiment, while the

fluorescence intensity recovers rapidly after bleaching (Figure 5.E;
magenta boxes), the original depolarized anisotropy value is eventually
recovered after a long delay of 4 minutes (Figure 5.F; magenta boxes).
Quantification of multiple such experiments at 37°C is presented in the
graph (Figure 5.F). These data suggest that at 37°C after a long
interval nanoclusters form from the monomers that have repopulated
the bleached area.
5.B.iv: Actin perturbation affects nano-cluster reformation –

further confirms nanoclusters are immobile
To confirm that anisotropy recovers at 37 °C due to formation of

clusters and not diffusion, the anisotropy nanocluster formation is
prevented. Such condition is achievable by treating cell with low level
of actin depolymerizing agent such as latrunculin (6μM). As we show in
the next chapter, there is a major redistribution of nano-cluster at the
cell surface if actin perturbing agents are used. Cells were treated with
6μM latrunculin for two minutes before starting the same ARAP assay,
where no significant morphological change can be visualized over time
period of the experiment. This resulted in an absence of the recovery of
anisotropy in the bleached spot at 37 °C over one or four minute time
points though intensity recovers and neighbouring pixels remain
depolarized as the membrane contains nanoclusters (Figure 5.G.i).
These data are consistent with the idea that while monomers are free
to diffuse, nanoclusters are immobile and formed in situ. The formation
of nanoclusters is sensitive to activity of cortical actin, which I will be
elaborating in the next chapter.
95
There is evidence in existing literature that the cortical actin

meshwork activity is related to myosin activity. Therefore, I next
examined whether a block in myosin activity could affect nonocluster
formation. To test this phenomenon in a flat membrane ARAP
experiment was performed after treatment with Blebistatin (50μM).
Similar result in ARAP experiment was obtained when treated when
Myosin activity is blocked (Figure 5.G.ii). These data again suggest
that replenishment of nanoclusters can only happen by formation of
new clusters from existing monomer and activity of acto-myosin
complex is needed for the process.
5.B.v: Formation of nanocluster is sensitive to levels of

cholesterol at the plasma membrane
Levels of cholesterol in the membrane play a important role ins

the formation of nanoclusters (Sharma et al., 2004; Varma and Mayor,
1998). Four minutes treatment with 10mM methyl β-cyclodextrin also
causes a reduction in the recovery of homo-FRET in the bleached
region on the cell surface in ARAP experiment at 37 °C (Figure 5.H).
Earlier work showed that low levels of cholesterol depletion leads to a
reduction of CA activity as observed by imaging the dynamics of CA
(Chadda et al., 2007), but not on the net nanocluster concentration
(Sharma et al., 2004). Similar result was found after four minutes of
treatment with methyl β-cyclodextrin where clusters still appear as
depolarized pixels as untreated cells. But these clusters do not diffuse
within the time period of experiment. While pre-formed cluster are
retained on the cell surface after pre-treatment with methyl β-
cyclodextrin, there is no de novo formation of cluster at the treated cell
surface. This data suggests that formation of nanocluster is extremely
sensitive to the levels of cholesterol in the membrane.
96
5.C: Discussion
The average picture obtained from previous studies of GPI-AP

organization (Sharma et al., 2004; Varma and Mayor, 1998) describes
presence of cholesterol sensitive sub-resolution (of fluorescence
microscope) clusters of the size of 2-4 molecules and 20-40% cluster
occupancy on the cell surface.
My studies described in this chapter regarding distribution of

clusters and its mobility on the cell surface suggest that there is a
preferential distribution of nanoclusters in specific regions of cell and
the distribution of nanoclusters in those regions is non-random. The
anisotropy recovery assay of nanoclusters shows that these clusters,

when bleached or in other words depleted, are immobile on the cell
surface and can only be reformed at physiological temperature from
mobile monomers. The monomeric population remains mobile at all
temperature. Since the clusters are recovering only at 37°C, the reason
behind this recovery is very much debatable. The explanation could
either be diffusion of clusters or replenishment by formation from
existing monomers. It is difficult to rule out the possibility of cluster
mobility and / or genesis. It is hard to believe that clusters are
transiently becoming mobile at 37°C unless they held by some
unknown entities at the plasma membrane specifically at non-
physiological temperature such as 20°C. Because, theory of simple
membrane diffusion cannot explain the fact that molecules embedded
into membrane and immobile at 20°C become mobile at 37°C only due
to temperature change. On the other hand, the time required to recover
the anisotropy at the bleached area is much highier compared to the
expected time needed to diffuse the clusters of size 2-4 molecules from
the neighboring area. Counter intuitively, it is possible that if I wait for
long time I will see a recovery even at 20°C, but then again I may see
some formation happening at this temperature and rate of which is
97
slower than 37°C. My explanation for new synthesis of clusters are
further supported by the fact that at 37°C, I see that there are active
component such actin and myosin are involved in this process. When
the fundamental molecular machines are inactivated by biochemical
agents I do not see recovery of clusters at depleted area (though they
are very much present in the neighboring area, they cannot diffuse).
Note that if the depletion area (bleaching area) at the cell surface is
very large or bleaching time is very long the anisotropy recovery assay
fails because of the overall depletion of the labeled monomers at the
membrane those will take part into the formation new clusters who can
still fluoresce and report to my assay.
98
5.D: References
Bacia, K., and P. Schwille. 2007. Fluorescence correlation

spectroscopy. Methods Mol Biol. 398:73-84.
Chadda, R., M. Howes, S. Plowman, J. Hancock, R. Parton, and S.
Mayor. 2007. Cholesterol sensitive Cdc42-activation regulates
actin polymerization for endocytosis via the GEEC pathway.
Traffic. in press.
Chen, Y., B.C. Lagerholm, B. Yang, and K. Jacobson. 2006. Methods
to measure the lateral diffusion of membrane lipids and proteins.
Methods. 39:147-53.
Fujiwara, T., K. Ritchie, H. Murakoshi, K. Jacobson, and A. Kusumi.

2002. Phospholipids undergo hop diffusion in
compartmentalized cell membrane. J Cell Biol. 157:1071-81.
Goswami, D., K. Gowrishankar, S. Bilgrami, S. Ghosh, R. Raghupathy,
R. Chadda, R. Vishwakarma, M. Rao, and S. Mayor. 2008.
Nanoclusters of GPI-anchored proteins are formed by cortical
actin-driven activity. Cell. 135:1085-97.
Kenworthy, A.K. 2007. Fluorescence recovery after photobleaching
studies of lipid rafts. Methods Mol Biol. 398:179-92.
Lommerse, P.H., H.P. Spaink, and T. Schmidt. 2004. In vivo plasma
membrane organization: results of biophysical approaches.
Biochim Biophys Acta. 1664:119-31.
Mayor, S., and M. Rao. 2004. Rafts: scale-dependent, active lipid
organization at the cell surface. Traffic. 5:231-40.
Rao, M., and S. Mayor. 2005. Use of Forster's resonance energy
transfer microscopy to study lipid rafts. Biochim Biophys Acta.
1746:221-33.
99
Varma, R., and S. Mayor. 1998. GPI-anchored proteins are organized in
submicron domains at the cell surface. Nature. 394:798-801.
100
Figure 5.A spatial distribution of nanoclusters
CHO cells expressing FR-GPI were labeled with PLBTMR and

fluorescence intensity (grey scale) and anisotropy images (pseudo-
coloured according to the indicated LUT) were imaged at 37°C (i) and
22°C (vi) on a line scanning confocal system. Anisotropy values from
isolated monomeric proteins (A∞) are indicated by a vertical line
(magenta) at the right of the LUT bar. Low anisotropy regions in
relatively constant intensity regions from flat regions of the cell shown
[box(ii,iii)], High anisotropy structures [box (iv,v)] correspond to tips of
lamellipodium, whereas the lamellum exhibits a low anisotropy. Scale
bar 8 µm.
101
Figure 5.B Statistical analysis of distribution of nanocluster
Plot of ln P(( A)) versus ( A − A ) A2 , derived from anisotropy
data from cell images as shown in the previous figure taken in confocal
microscope. This shows a slower, exponentially decaying tail for FR-
GPI-expressing cells, which appears as a linear decay in the log plot
(black line).
102
Figure 5.C Anisotropy Recovery After Photobleaching at 20°C images
Fluorescence intensity (grey scale) and anisotropy (pseudo-

coloured) images of PLBTMR–labeled cells were recorded on line
scanning confocal microscope at 20°C , prior to (Pre-Bleach),
immediately-post (Bleach, intensity only) or after 1 or 4 min of (Post-
Bleach, 1 or 4 min, respectively) bleaching the region outlined in the
magenta box. Average anisotropy values from the bleached (magenta)
and unbleached (blue, brown) boxes are shown below pseudo-
coloured anisotropy images from each coloured-box.
103
Figure 5.D Graph shows quantification of ARAP data at 20°C
Graphs shows normalized fluorescence intensity (lower panel)

and average (and standard error) anisotropy values (upper panel) from
the respective coloured boxes under the time points indicated on the x-
axis, derived from measurements made on multiple cells (n≥6) at 20°C.
104
Figure 5.E Anisotropy Recovery After Photobleaching at 37°C images
Fluorescence intensity (grey scale) and anisotropy (pseudo-

coloured) images of PLBTMR–labeled cells were recorded on line
scanning confocal microscope at 37°C , prior to (Pre-Bleach),
immediately-post (Bleach, intensity only) or after 1 or 4 min of (Post-
Bleach, 1 or 4 min, respectively) bleaching the region outlined in the
magenta box. Average anisotropy values from the bleached (magenta)
and unbleached (blue, brown) boxes are shown below pseudo-
coloured anisotropy images from each coloured-box.
105
Figure 5.F Graph shows quantification of ARAP data at 37°C
Graphs shows normalized fluorescence intensity (lower panel)

the respective coloured boxes under the time points indicated on the x-
axis, derived from measurements made on multiple cells (n≥6) at 37°C.
106
Figure 5.G Graph shows quantification of ARAP data after latrunculin
and blebbistatin treatment
Graphs show normalized fluorescence intensity (lower panel)

an ARAP experiment as described in Figure 5.E, carried out at 37°C on
PLBTMR-labeled FR-GPI expressing cells, pre-treated for 4 min with Lat
( 6 µM) or blebbistatin (F; 50 µM) at 37°C. Magenta symbols represent
data obtained from the area subject to photo-bleaching while blue and
brown symbols are obtained from corresponding neighbouring areas.
Data were derived from measurements made on multiple cells (n≥8) in
a single experiment and averaged over at least two independent
experiments.
107
Figure 5.H Graph shows quantification of ARAP data after methyl β-
cyclodextrin
Graphs show normalized fluorescence intensity (lower panel)

and average (and standard error) of anisotropy values (upper panel)
from an ARAP experiment as described in Figure 5.E, carried out at
37°C on PLBTMR-labeled FR-GPI expressing cells, pre-treated with
mβCD (10 mM; 4 min). Magenta symbols represent data obtained from
the area subject to photo-bleaching while blue and brown symbols are
obtained from corresponding neighbouring areas. Data were derived
from measurements made on multiple cells (n≥6) in two independent
experiments.
108
Chapter 6
Cortical actin driven steady state dynamics of

GPI-AP monomers and nanoclusters – an active
process
6.A: Introduction
Dynamics of membrane anchored proteins is a topic of

research for a long time. Researchers have used different techniques
to study association and dissociation kinetics of membrane proteins at
the cell surface. These include Fluorescence Recovery After

Photobleaching (FRAP), Fluorescence Correlation Spectroscopy
(FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), dual-
color single molecule dynamics, and a few others. It has been noticed
that across different groups, results from these experiments vary
depending on the modes of observation. Although, detecting diffusion
of membrane anchored molecules is comparatively easy using various
biophysical tools, aggregation kinetics measurement remains a difficult
task. Some of reasons are as follows: – a) little control over the
concentration of reactant and product; b) time lapse observation cannot
be conducted always, since the system is at some steady state; c)
chemical composition of membrane, which is acting as the platform for
aggregation kinetics, may vary locally; d) the read out of association
and dissociation is never unique and quantitative.
In this chapter I focus my attention on the dynamics of GPI-

anchored protein clustering at the cell surface.
109
Nanocluster dynamics II
6.B: Results
6.B.i: Assay to study steady-state dynamics of GPI-AP

organization at the cell surface
I continue to explore the steady state dynamics of the GPI-AP

monomers and nanoclusters in the nanocluster-rich flat part of cell. In
the previous chapter, I have already described how temperature
influences reorganization of GPI-AP nano-cluster at the cell surface
using a protocol called ARAP. Here, I developed a novel fluorescence
assay, similar to a microphotolysis type assay (Peters et al., 1981),
where a simultaneously intensity and anisotropy trace (FIAT) is

collected from a confocal volume on cell surface (Figure 6.A.i; red
cross) illuminated with a multi-photon laser couple to a single photon
counting device (Figure 4.A). In this assay, I locally perturb the
distribution of PLF-labeled folate receptor (FR-GPI) at the surface of
CHO cells by multi-photon (MP) confocal excitation starting at time t=t0
up to t=t1 which I specify as first illumination period; then the laser is
switched off for a waiting time, tw; after this I switch on again for t2 time,
referred as second illumination period (Figure 6.A.ii). I follow the
dynamical response in fluorescence anisotropy and fluorescence
intensity from the same volume, where intensity represents the local
concentration of the protein and anisotropy represents the
oligomerization status of these receptors.
6.B.ii: Lipid shows typical concentration dependent FRET

signal
As a control, the cell membrane was labeled with an

exogenously added fluorescent lipid, BODIPY-SM, at concentrations
high enough to record significant homoFRET. The same recovery
110
assay was performed on the cell surface at 20 °C. However, during the
waiting time (tw), the anisotropy of BODIPY-SM recovers to its original
value when the intensity of the probe recovers substantially during this
time interval (Figure 6.B). This result suggests that a cell surface
molecule (largely on the outer leaflet of the plasma membrane) such as
BODIPY-SM, capable of unhindered diffusion (Klein et al., 2003),
recovers its intensity and anisotropy (and hence its original steady
state distribution) following localized photobleaching.
6.B.iii: GPI-AP nanoclusters remain immobile at the scale of

confocal area and follows unusual interconversion.
During the first illumination, the time trace of fluorescence

emission intensity shows an initial rapid loss followed by a slower
decay, and significant recovery during tw (Figure 6.C; blue dots). The
corresponding emission anisotropy trace, broadly, shows two kinds of
behaviour that appears correlated with temperature. At 20°C, the initial
anisotropy in the illuminated volume is depolarized (Figure 6.C; red
line), characteristic of a flat membrane where GPI anchored protein
remains as mixture of nanoclusters and monomers. During the first
illumination period (t1) there is a sharp initial rise in fluorescence
anisotropy (corresponding to rapid loss of homoFRET), before
saturating to a high value, due to fluorophores being bleached
continuously. Then the anisotropy value reaches the values
corresponding to the isolated monomers in the membrane (A∞; Figure
6.C, pink band). On the other hand, at 37 °C (Figure 6.D; red line), the
anisotropy rises during t1, as the fluorophores in the confocal volume
get bleached, and saturates to a value significantly lower than A∞.
The recovery of fluorescence intensity in the observation volume

depends on the durations, t1 and tw and this is independent of
111
temperature. If t1 is small (<20s) and tw large (>30s), the fluorescence
intensity recovers significantly, implying that fluorophores diffuse in
from the surrounding regions. However, the fluorescence anisotropy at
20 °C, starts out with the same saturation value obtained at the end of
the first illumination, and does not recover to that expected of the
original mixture of nanoclusters and monomers (Figure 6.C, red line).
This implies that nanoclusters neither reform within, nor are
replenished from the reservoir of unbleached fluorophores present
outside the illuminated volume. In contrast, at 37 °C, there is an almost
complete restoration of the original depolarized anisotropy value after
tw, implying that there is substantial reassembly of nanoclusters from
monomers at 37 °C (Figure 6.D, red line).
6.B.iv: Temperature dependence of the dynamics
The dynamical response in FIAT assay (detailed in Figure 6.A)

is systematically recorded from different flat regions of cells, at
temperatures ranging from 15°C–37°C. The fluorescence intensity and
anisotropy profile in each FIAT assay was modeled by reaction-
diffusion type equations (Figure 6.F; this work was done in
collaboration with Kripa G. and Madan Rao; (Goswami et al., 2008)),
incorporating multiple parameters: diffusion of monomers and
nanoclusters (diffusion coefficients, D1 and Dc), bleaching of
fluorophores (bleach rate, b), and the interconversion between
monomers and nanoclusters (aggregation and fragmentation rates, ka
and kf). Using the anisotropy values for monomer and nanocluster, Am
and Ac, we can fit the data to extract these parameters assuming the
fraction of nanoclusters having n proteins, m of which are unbleached,
present within the confocal volume at time, t.
112
Using the model, we fit the calculated intensity and anisotropy
profiles to the experimental data at different temperatures and extract
the best fit values for the parameters (Figure 6.G). The diffusion
coefficient of the nanoclusters Dc, obtained from the fit, is found to be
negligibly small (Dc ≈ 0) at all temperatures – this suggests that while
the monomers are mobile (Figure 6.H), the nanoclusters are immobile,
consistent with data shown in previous chapter.
6.B.v: Spatial heterogeneous nature of association-

dissociation kinetics at the cell surface irrespective of
temperature.
Interconversion parameters at every temperature, extracted by

fitting intensity-anisotropy traces, obtained are spatially variable on the
cell surface. This corroborates the heterogeneity in the steady state
distribution of nanoclusters at the cell surface. Same assay has been
performed on randomly chosen points on multiple cell surface and
analysis showed variation in the rates of association and dissociation
kinetics. Although the interconversion rates are biased for different
temperatures, a distribution of formation kinetics was observed for
every temperature (Figure 6.I).
6.B.vi: Non-Arrhenius interconversion dynamic
At a given temperature, the data for interconversion kinetics

parameters cluster can be divided into four qualitatively distinct
classes: Full Recovery (FR), Partial Recovery (PR), No Recovery (NR)
and No Interconversion (NI) (schematic in Figure 6.E). Interconversion
dynamics is typically absent at lower temperatures and present at
higher temperatures (Figure 6.J). According to Figure 6.G, these
classes reflect the spatial heterogeneity in the organization of
113
nanoclusters in the flat regions of the cell surface. Representative class
for each temperature (demarcated in red in Figure 6.I and 6.J.i) was
chosen to construct an Arrhenius-plot from the typical value in each
representative class as a function of the inverse temperature (Figure
6.J.ii); the curve is almost flat at temperatures above 24 °C and
changes sharply below this temperature. This chemical reaction does
not follow a typical Arrhenius behavior.
6.B.vii: Cholesterol-sensitive interconversion
Previousl studies have shown that the steady state distribution

and nanoclustering is sensitive to cholesterol levels in the membrane

(Sharma et al., 2004). Therefore, I tested if the dynamics is affected by
an acute perturbation of cholesterol levels. I conducted similar FIAT
experiments at 37°C with a mild treatment of mβCD (10mM) 30
minutes prior to the first illumination. Whereas on control cells, the
cluster concentration recovers partially or completely after a time t1+tw
(Figure 6.D), I observed that application of mβCD prior to the first
illumination prevents the restoration of the original depolarized
anisotropy value during the waiting period, although the fluorescence
intensity has completely recovered (Figure 6.K). Thus, these results
suggest that the interconversion dynamics is sensitive to cholesterol
levels in the membrane.
6.B.viii: Role of cortical actin in interconversion
Cortical actin activity was perturbed using Jasplakinolide (Jas)

or Latrunculin (Lat) at 37°C to determine if actin polymerization activity
is necessary for maintenance of nanoclusters at the plasma
membrane. Prolonged incubation with Jas / Lat results in the
114
generation of micron-sized blebs, devoid of CA (Figure 6.L). Time-
resolved fluorescence measurements show that these fully-formed
blebs lack GPI-AP nanoclusters. Donor fluorescence lifetime obtained
from PLF (donor) and acceptor (PLR) labeled blebs are higher
compared to the flat parts (cluster rich regions) of cells and matches
with the donor alone lifetime data (Figure 6.M; Table I). Fluorescence
lifetime images show (Figure 6.N) blebs with comparatively higher
donor fluorescence lifetime (pseudo-color coded) compared to the flat
region of the cell. Increase in fluorescence lifetime both single decays
and images explain that the hetero-FRET between donor-acceptor
labeled GPI-anchored protein has decreased. The proximity between
molecules in such organization is not present anymore when the
membrane integrity is lost in bleb, which is also devoid of cortical actin.
The steady-state anisotropy values indicate whether the

anisotropy value is integrated with the FRET and rotational component
of the fluorophore. It is always questionable about the origin of the
polarized or depolarized values obtained in the steady-state anisotropy
measurements. But in time resolved anisotropy decay measurements
the fast anisotropy decay component specifically reports the amount of
homo-FRET present in the system. The rate of decay further reports
about the distance between the fluorophore those are in FRET
proximity. Time resolved anisotropy data from blebs, generated from
GFP-GPI expressing cells, showed no or less in amplitude of fast
decay (homo-FRET) component (Figure 6.O and Table II). Since blebs
lack fast decay component or the amplitude is less than obtained from
the flat part of cell, it is confirmed that GFP-GPI organization is lost in
blebs. It is also noticeable that decay data (in case of latrunculin
generated blebs) from blebs having less amount of fast component has
much higher decay rate. This suggests that in latrunculin generated
blebs the GFP-GPI molecules are further separated compared to
115
normal organization. It possible that in these blebs few organized
molecules are left with increased intermolecular distance either
because of incomplete block or re-distribute molecules in a cluster too
some extent.
This is again confirmed by the polarized value of anisotropy in

FIAT assay. Polarized value of anisotropy remains unchanged in the
range of A∞ through out the period of illumination (Figure 6.P; red line)
suggesting bleb devoid of nanoclusters.
Next I applied low concentrations of Jas or Lat on cells (5 and 6

µM respectively), which does not cause any morphological change in
cells within the observation time of the experiment, to explore the

status of the dynamics of nanoclusters. Whereas, the nanocluster
concentration recovers completely after time t1+tw on control cells at
37°C (Figure 6.Q, red line), when low concentration of Lat or Jas has
applied either in the beginning or during waiting period prevents the
restoration of the original depolarized anisotropy value (Figure 6.Q.i
and ii; red lines), although the fluorescence intensity has completely
recovered (Figure 6.Q.i and ii; blue lines). I have also shown that pre-
treatment with Lat affect the dynamics of nanoclusters at the plasma
membrane due to dynamic polymerization / depolymerization of actin
near the membrane is restricted (Figure 6.Q.iii). Earlier it had been
shown that treatment with blebbistatin, which restricts the myosin
activity, stops bleb retraction process and nanocluster cannot reform at
the same site after the bleb retracts back to the membrane (Charras et
al., 2007; Charras et al., 2006; Goswami et al., 2008; Keller et al.,
2002). Therefore, I examined the effect of perturbation of myosin
activity using blebbistatin (50µM), on dynamics of interconversion at
the flat membrane (Figure 6.R; red trace). Whereas, at 37°C in
presence of blebbistatin, neither there is de novo synthesis of
116
nanoclusters of GPI-APs nor do they diffuse in from the neighboring flat
membrane area. Altogether, these results suggest that the nanocluster
intercoversion dynamics is linked with acto-myosin activity by some
unknown mechanism near the cell surface and nanoclusters are
immobile even at 37°C on the cell surface.
6.C Discussion
The experimental evidence detailed here show that the

dynamics of formation and fragmentation is dependent on the local
concentration of cholesterol and CA remodeling. Salient features of this
unusual kinetics behavior are: (i) nanoclusters are immobile; (ii)

formation of nanoclusters are temperature sensitive (also includes non-
Arrheneius type of chemical reaction with a cross-over at 24°C) and
violates law of mass action; (iii) spatial variation of interconversion
dynamics of nanoclusters due to spatial variation in levels of active
CA; (iv) involvement of actin and myosin activity on interconversion
dynamics (acto-myosin activity has also been reported to show a sharp
crossover at ~24°C); (v) above 28°C, the calculated binding energy of
nanoclusters is ∆E/kBT≈10-2 and this is 2-3 orders of magnitude lower
than the minimal (van der Waals) interactions between molecules on a
membrane at a similar intermolecular distance.
117
6.D: References
Charras, G.T., M. Coughlin, T.J. Mitchison, and L. Mahadevan. 2007.

Life and Times of a Cellular Bleb. Biophys.
J.:biophysj.107.113605.
Charras, G.T., C.K. Hu, M. Coughlin, and T.J. Mitchison. 2006.
Reassembly of contractile actin cortex in cell blebs. J Cell Biol.
175:477-90.

Keller, H., P. Rentsch, and J. Hagmann. 2002. Differences in cortical
actin structure and dynamics document that different types of
blebs are formed by distinct mechanisms. Exp Cell Res.
277:161-72.
Klein, C., T. Pillot, J. Chambaz, and B. Drouet. 2003. Determination of
plasma membrane fluidity with a fluorescent analogue of
sphingomyelin by FRAP measurement using a standard
confocal microscope. Brain Res Brain Res Protoc. 11:46-51.
Peters, R., A. Brunger, and K. Schulten. 1981. Continuous
fluorescence microphotolysis: A sensitive method for study of
diffusion processes in single cells. Proc Natl Acad Sci U S A.
78:962-966.
Volkmer, A., V. Subramaniam, D.J. Birch, and T.M. Jovin. 2000. One-
and two-photon excited fluorescence lifetimes and anisotropy
decays of green fluorescent proteins. Biophys J. 78:1589-98.
118
Table I: Hetero-FRET+ measurement by Donor Fluorescence Lifetime
Fluorescence Lifetime§§ of PLF-labeled FR-GPI at the surface of

FR-GPI-expressing CHO cells
D A
Treatment #
τavg †† (ns)
(Donor) (Acceptor)
Control 2.28
PLF 10
(Flat regions) @ (±0.04)
Control 2.00
PLF PLR 11
(Flat regions) @ (±0.13)
Saponin 0.2% 2.30

PLF PLR 5
(±0.09)
Latrunculin 2.32
PLF PLR 5
(Bleb)* (±0.09)
Jasplakinolide 2.16
PLF PLR 5
(Bleb)* (±0.01)
In all experiments CHO cells-expressing FR-GPI were singly

labeled with donor (D) alone (PLF, 160nM) or with donor (D) and
acceptor (A) fluorophores (PLF, 160 nM; PLR, 200 nM). Time
correlated single photon statistics after excitation with multi-photon
excitation were obtained as described in Chapter 3.
+Efficiency of hetero-FRET in control cells is estimated by

comparing the average lifetimes of PLF in the presence and absence
of the acceptor fluorophore (PLR); shorter lifetime in the presence of
acceptor indicates increased FRET.
Note: Cholesterol removal by saponin-treatment increases the
lifetime of donor PLF to that obtained in the absence of the acceptor.
119
PLF/PLR-labeled blebs exhibit a longer donor-lifetime, consistent with
the lack of hetero-FRET on the blebs.
§§
Fluorescence lifetimes were calculated using the fitting routine
outlined in Supplementary Methods and expressed as averages (+
S.D.) from the indicated number of cells (n #).
††
τavg is the amplitude-weighted average of all lifetimes obtained
from the fitting routine; PLF in the absence of acceptor shows multiple
lifetimes as reported earlier(Sharma et al., 2004).
@
Measurements on flat regions were made by scanning the
laser over a small ~100x100 pixel area.

* Measurements on Blebs were made by collecting photon
statistics using a parked beam located on the bleb.
120
Table II: Time resolved Homo-FRET measurements
Anisotropy decay rates# of GFP-GPI at the surface of GFP-GPI-

expressing CHO cells
Treatment #
r0* τ1 (ns) a1 τ2 (ns) a2 rss
Control 0.40 0.17 0.10 33 0.90 0.34

11
(Flat regions) (± 0.01) (± 0.06) (± 0.01) (± 11) (± 0.01) (± 0.02)
Saponin 0.39 23 0.36
9
0.2% (± 0.02) (± 4) (± 0.02)
Latrunculin 0.40 0.2 0.08 27 0.92 0.34

7
(Flat regions) (± 0.01) (±0.06) (± 0.04) (± 7) (± 0.04) (± 0.02)
Jasplakinolide 0.41 0.3 0.09 28 0.92 0.35
7
(Flat regions) (± 0.01) (±0.02) (± 0.05) (± 9) (± 0.05) (± 0.03)
Latrunculin 0.41 0.57 0.06 42 0.94 0.38
6
(Bleb) (± 0.01) (± 0.07) (± 0.01) (± 22) (± 0.006) (± 0.01)
Jasplakinolide 0.41 39 0.39
6
(Bleb) (± 0.01) (± 13) (± 0.01)
In all experiments CHO cells-expressing GFP-GPI were placed

on the microscope stage and the laser beam was parked on indicated
regions. Time correlated single photon statistics were obtained after
excitation with multi-photon excitation using a high NA objective as
described in Supplementary Methods. Measurements on all regions
were made by collecting photon statistics using a parked beam located
on the region.
Note: Cholesterol removal by saponin-treatment eliminates the

fast component due to FRET.
121
+Efficiency of homoFRET is obtained by estimating the

amplitude of the fast decay component of the anisotropy decay of GFP
fluorescence emission. While the rate of decay of the short component
is an estimate of the distance between GFP-fluorophores, the longer
decay component relates to the rotational dynamics of the GFP-
fluorophore.
Note: Cholesterol removal by saponin-treatment eliminates the

fast component consistent with earlier studies. Anisotropy decay rates
on blebs also have much smaller amplitude of the shorter decay rate
(Lat) or no short component (Jas), consistent with a reduction/lack of
homoFRET between GFP-GPI-APs in these regions. The presence of

this small component is likely to be due to the large volume of the
confocal excitation (~ 900 nm, Z-resolution), potentially collecting some
emission from the flat-regions surrounding the blebs.
* r0 is the initial anisotropy; its value is depolarized compared to

that obtained using a low NA objective as reported earlier (Volkmer et
al., 2000) (data not shown).
# Anisotropy decay rates were calculated using the fitting routine

eplained in chapter 3 and expressed as averages (+ S.D.) from the
indicated number of cells (n).
122
Figure 6.A Imaging and interconversion dynamics assay
PLF-labeled FR-GPI expressing cell imaged on Zeiss 510 meta

microscope (Zeiss, Germany) illuminated by multi-photon excitation at
790 nm. ii) Schematic representation of the strategy of FIAT or
microphotolysis assay developed to study the interconverion of
nanocluster of GPI-APs at the cell surface. Intensity and anisotropy
traces from a fluorescent sample were collected from a multi-photon
excitation volume focused on the sample plane. This was achieved
with a 20x - objective lens (0.7NA). The multiphoton laser was parked
at a single point (as shown by the red crosshair) for continuous
illumination at or near the cell periphery at the center of the field of
observation. Scale bar, 5 µm.
123
Figure 6.B FIAT assay for BODIPY-SM at cell surface
BODIPY-SM (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-
indacene-3-pentanoyl)) complex with BSA (ratio 1:1) in a 5 µM solution
was incorporated onto the surface of CHO cells by incubating for 30
min on ice. Cells were subsequently maintained at 20°C on a Laser
scanning confocal microscope equipped with MP-excitation (790 nm).
Cells were imaged using MP excitation (top) and intensity (blue line)
and anisotropy traces (red line) were obtained simultaneously from a
confocal volume (red crosshair) during an illumination sequence
outlined at the top of the trace (bottom). The concentration of BODIPY-
SM incorporated on the cell surface, is sufficient to record significant
homo-FRET at time, t=0. During the initial illumination period (t1), the
anisotropy of BODIPY-SM increased and approached A∞. A∞ (pink
band) was measured after photobleaching the BODIPY-SM so that
there was no further change in anisotropy. Scale bar 6.6µm.
124
Figure 6.C Intensity and anisotropy traces and images from cell
surface-labeled GPI-APs at 20°C.
Intensity (blue line) and anisotropy (red line) traces were

obtained simultaneously from the resultant confocal volume [e.g. red
crosshair, inset in cell image], during the illumination sequence outlined
at the top. The pink band in the graphs are the range of A∞ values
obtained for each experiment representing anisotropy values for PLF
labeled monomeric GPI-anchored protein.
125
Figure 6.D Intensity and anisotropy traces and images from cell
surface-labeled GPI-APs at 37°C.
Examples of intensity (blue line) and anisotropy (red line) traces

which were obtained simultaneously from cell surface at 37°C in a
confocal volume during FIAT experiments as the illumination sequence
outlined at the top. The pink band in the graphs are the range of A∞
values obtained for each experiment representing anisotropy values for
PLF labeled monomeric GPI-anchored protein at 37°C.
126
Figure 6.E Schematic representation of FIAT assay on cell surface
In this figure, I describe the schematic of various types of

fluorescence intensity and anisotropy traces obtained from FIAT assay
of GPI-APs organization in a confocal volume at the cell surface. A
pictorial description of classification made by visual inspection of
graphs obtained from multiple experiments: Full recovery (FR) and
partial recovery (PR) types would be expected from a region where
there are more nanoclusters and relatively significant monomer-
nanocluster interconversions occur. No recovery type (NR) is expected
from regions where significant fragmentation and no formation of
nanocluster occur. No inerconversion (NI) is type of traces where
monomeric population of GPI-APs is not engaged in formation of

nanocluster.
127
Figure 6.F Schematic of dynamics of GPI-anchored proteins
Schematic of the expected dynamics (bleaching, diffusion and

interconversion) of monomers and nanoclusters (depicted as dimers)
within a confocal volume (inner circle). The colour of the circles
represents the bleaching status of the fluorophores, green being active
and black bleached. The arrows represent the transition rates between
the species present. While the fluorophores diffuse both into and out of
the confocal volume, the model assumes that the incoming
fluorophores are predominantly unbleached. The outer circle
represents the pool of unbleached monomers and nanoclusters that
serves to replenish the confocal volume. The dynamics is modeled by
reaction-diffusion equations, involving rates of bleaching (b), diffusion

(monomer d1 and cluster dc), and cluster aggregation (ka) and
fragmentation (kf) as described.
128
Figure 6.G Examples of theoretical fits obtained from FIAT assay
Examples of model dependent fit obtained from the schematic

described in previous figure of FIAT experiments of PLF-labeled FR-
GPI, recorded at different temperatures 37°C (i), 24°C (ii) and 15°C (iii,
iv). The fits (dark lines) provide the values of the parameters appearing
on top of each panel. Extracted values of dc were found to lie in the
range (-10-5 s-1 < dc < 10-5 s-1); this being 4-orders of magnitude smaller
than d1. This may safely be taken as immobilized clusters. Pink band
represents the A∞ values.
129
Figure 6.H Rate of diffusion of monomers obtained from the fit
Diffusion of monomers of FR-GPI d1 measured across different
cells as a function of temperature. The range of values for d1 is shown
normalized to a typical value of FR-GPI diffusion (0.1078s-1) extracted

at 37oC. This is consistent with those obtained from measurements on
FR-GPI on similar blebs and BODIPY-SM on the cell surface. In
addition, independently measured variation of diffusion coefficients
from FCS measurements of FR-GPI and GFP-GPI across the
temperature range are 0.516 ± 0.135 µm2/s (20°C) and 1.276 ± 0.469
µm2/s (37°C).
130
Figure 6.I Interconversion rates obtained from the fit
Graph shows the rates of fragmentation ( k f ) versus aggregation
( ka ) measured across different cells at multiple temperatures.
Distribution of the measured parameters was observed upon fitting,

across different cells at temperatures ranging from 15°C - 37°C.
The ranges of data that falls into these categories FR, PR and NR
classification was segmented with dashed lines correspond to the
classes elaborated previously.

131
Figure 6.J Temperature dependence of interconversion rates
i) Histogram shows the relative population of each class [FR,

PR, NR, and NI (where the anisotropy stays at a highly polarized value
A∞ at all times)] at different temperatures based on the classification
scheme (Figure 6.E); the representative class for each temperature is
indicated by the red bar. ii) Ratio of typical interconversion rates
obtained from each representative class are represented in an
Arrhenius plot, as ln ka/kf versus Troom T . The line connecting the
typical values of ln ka/kf in each representative class shows strong non-

Arrhenius behaviour.
132
Figure 6.K Perturbation of Cholesterol levels in membrane alters
dynamics
PLF-labeled FR-GPI expressing cells were pre-incubated with

mβCD (10 mM; 30 min) on a microscope stage maintained at 37°C,
were illuminated by multi-photon excitation at 790 nm. Intensity (blue
line) and anisotropy (red line) traces were obtained simultaneously
from the resultant confocal volume, during the illumination sequence
outlined at the top. After waiting time tw, the traces were recorded from
the same area of the cell during t2. The pink band at the top indicates
the range of A∞ values obtained in the experiment.
133
Figure 6.L Blebs are devoid of actin
GFP-GPI expressing cells were treated with Jas (14µM) for

30min at 37 °C, fixed and stained with rhodamine phalloidin, before
imaging on confocal. Images from the bottom, medial and top planes of
a confocal stack of images show that membrane blebs (green) are
devoid of polymerized actin (red) as observed by the lack of
Rhodamine-phalloidin staining. Scale bar, 6 µm.
134
Figure 6.M Hetero-FRET measurements by donor fluorescence
lifetime: Blebs are devoid of nanoclusters
FR-GPI expressing CHO cells were labeled with donor and

acceptor (PLF and PLR) cells were imaged using the multiphoton set
up after treatment with Jas for 30 min at 37 °C. The bleb and flat part of
membrane are marked with green box and blue box respectively in the
intensity image. Lower donor lifetimes (corresponding to high levels of
heteroFRET) are observed in the flat regions (blue graph) of cells while
higher donor lifetimes representing less FRET were recorded from the
blebs (green graph). Statistics of lifetime data from large set of data is
represented in Table I. Scale bar, 20 µm.
135
Figure 6.N FLIM data shows blebs has less hetero-FRET compared to
flat part of cell
Either donor (PLF) alone (panel i) or donor and acceptor

(PLF/PLR)-labeled FR-GPI expressing CHO (panel i, iii) cells were
imaged using the FLIM set up either before (panel i, ii) or after
treatment with Jas for 30 min at 37 °C (panel iii). Average donor
fluorescence lifetime image pixel-by-pixel were generated for specific
regions of the cell demarcated by boxes at the cell surface at 1 x 1 µm2
resolution. This was done to achieve the the photon statistic required
for the calculation of donor lifetime. The pseudo-coloured life-time
maps at the right of each intensity image, coloured with respect to the
LUT scale corresponding to the indicated life-time ranges. The spatial

resolution of PLF-fluorescence at signal levels obtained is not sufficient
to resolve the micron-scale features. However, the spatial variation of
heteroFRET signal estimated from the measured distribution of
average fluorophore lifetimes compared to that observed for donor-
alone labeled cells [panel i, boxes (1-6)], reveals regions with high
FRET [panel ii, flat regions in boxes (1-3, 5)] and low FRET [panel ii,
membrane ruffles in boxes (4, 6);] and [panel iii, blebs in boxes (1-6)].
Donor lifetime decay data was also obtained from flat part of cell and
blebs by scanning or parking the laser at a single spot. This set of data
is summarized in Table I.
136
Figure 6.O Time-resolved anisotropy data for homo-FRET
measurement
GFP-GPI-expressing CHO cells were treated with Lat A (25 µM,

30 min, 37 °C) to generate blebs, and at the indicated spot on a bleb
(green cross), single point-time resolved anisotropy decay
measurements was carried out using confocal multi-photon excitation
at 37°C. The anisotropy decay profiles obtained from flat region (blue
arrow; blue line) are compared with those blebs (green crosshair;
green line) of cell in the same image. Data from multiple flat
membranes, blebs and from cell membranes treated with saponin are
shown in Table II. Scale bar, 20 µm.
137
Figure 6.P Nanoclusters are not present in membrane structure devoid
of actin (blebs) as seen in FIAT experiments
FIAT experiment was performed on blebs generated by 30 min

incubation at 37 °C with Lat (12 µM; B) or Jas (15 µM; C) show high
anisotropy values close to A∞ (pink band) indicating membranes devoid
of pre-existing GPI-AP nanoclusters. Intensity (blue) and anisotropy
(red) traces are shown.
138
Figure 6.Q Actin perturbation influences nanocluster formation
FIAT experiment was performed on PLF-labeled FR-GPI

expressing cells. With same from the illumination sequence intensity
(blue) and anisotropy (red) traces were obtained at 37°C where
Latrunculin (Lat, 6µM; i) or Jasplakinolide (Jas, 5µM; ii) was added to
cells on the stage after the first illumination period, t1, or pre-incubated
with Latrunculin (Lat, 6µM; iii) for 4 minutes. After waiting time tw, the
traces were recorded from the same area of the cell during t2. During
this treatment there was no detectable change in cell morphology.
139
Figure 6.R Inhibition of myosin activity influences nanocluster formation
FR-GPI expressing cells labeled with PLF were pre-incubated

with Blebbistatin (50µM) for 4 minutes on microscope stage at 37°C
and FIAT experiments were performed. Intensity (blue line) and
anisotropy (red line) traces were obtained simultaneously from the
resultant confocal volume, during the illumination sequence outlined at
the top. The pink band at the top of each panel indicates the range of
A∞ values obtained in the experiment. It is evident that when myosin
activity is blocked even at 37°C no further formation of nanoclusters
after depletion by bleaching in a confocal area. During this treatment
there was no detectable change in cell morphology.
140
Chapter 7
Conclusions and discussion
Conclusions:
I summarize the conclusions of my thesis in following points:
1. I have estimated energy transfer efficiency in GPI-AP

organization by hetero-FRET method with varying labeling
ratios of acceptor and donor fluorescent probe. The profile

of the energy transfer efficiency is similar to the theoretical
prediction.
2. I have also shown that both homo- and hetero-FRET
measurement are complementary technique to estimate
altered oligomerization status of GPI-APs obtained upon
generating larger scale oligomer. In general, these
techniques together can be reliably used to determine scale
of molecular organization at the nanometer scale.
3. Both ARAP and FIAT data and analysis show that at the
cell surface nanoclusters are immobile at different length
scale (from confocal area to 1μm2), but monomers remain
mobile.
4. Nanoclusters of GPI-AP are formed at physiological
temperature from existing monomers and maintained at a
specific concentration irrespective of the amount of the
protein present at plasma membrane. Therefore, this
system remains un-equilibrated.
5. I measure the rate of formation and fragmentation of
nanoclusters in microphotolysis type FIAT assay at multiple
141
Conclusions
temperatures. In collaboration with Kripa G. and Dr. Madan
Rao, we show the interconversion kinetics between
monomer and clusters do not exhibit Arrhenius type
behavior.
6. Finally, I show that the formation of nanoclusters is
sensitive to the membrane composition (levels of
cholesterol) and cortical actin activity.
Discussion:
The formation and immobilization of native nanoclusters of GPI-

APs are created and maintained by the CA (Goswami et al., 2008). It

represents a new type of molecular complexation in steady-state
kinetics of lipd-anchored protein at the live cell surface. This can be
explained through a framework of active hydrodynamics of the
membrane which is coupled to active cytoskeleton system (Hatwalne et
al., 2004; Kruse et al., 2004; Manville et al., 2001; Ramaswamy and
Rao, 2007). Here, passive cell surface molecules, such as GPI-APs or
transmembrane proteins, irrespective of their direct or indirect
interactions with the CA, can transiently be influenced by the CA.
Theoretical studies speculate such transiently bound molecules can be
actively driven along the polar actin filaments resulting in local
molecular clustering and can further be explained by acto-myosin
contractile forces and tread-milling. These active complexes maybe a
generic mechanism for local nanoclustering of a variety of cell surface
molecules such as GPI-APs (Sharma et al., 2004), Ras-isoforms,
(Plowman et al., 2005), and gangliosides (Fujita et al., 2007). These
nano-complexes can increase the rates of chemical reactions in living
cells (Hancock, 2006). The larger scale (> 100 nm) organization of
these nanoclusters maintained by CA, could possibly be act as
142
Conclusions
functional membrane domains responsible for signaling and sorting
functions (Sharma et al., 2004).
The concept of lipid-based sorting and signaling by lateral

segregation of specific molecules as a part of ‘lipid-rafts’ can be
implemented as following (Simons and Ikonen, 1997): I show that lipid
based clustering of molecules may be achieved by active mechanisms
followed by concentration of such nano-domains into specific sites.
These sites can then act as hot-spots for endocytosis and signalling.
This has implications in understanding the role of rafts, especially as
regulatable membrane microdomains as envisaged by recent studies in
cell signalling networks involved in regulating cell shape (Neves et al.,
2008). It is also likely that such structures may exhibit distinct

properties, quite distinct from those dictated by thermodynamic
considerations as explored in artificial membranes.
References:
Fujita, A., J. Cheng, M. Hirakawa, K. Furukawa, S. Kusunoki, and T.

Fujimoto. 2007. Gangliosides GM1 and GM3 in the living cell
membrane form clusters susceptible to cholesterol depletion and
chilling. Mol Biol Cell. 18:2112-22.
Hancock, J.F. 2006. Lipid rafts: contentious only from simplistic
standpoints. Nat Rev Mol Cell Biol. 7:456-62.
Hatwalne, Y., S. Ramaswamy, M. Rao, and R.A. Simha. 2004.
Rheology of active-particle suspensions. Phys. Rev. Lett. .
92:118101.
143
Conclusions
Kruse, K., J.F. Joanny, F. Jülicher, J. Prost, and K. Sekimoto. 2004.
Asters, vortices and rotating spirals in active gels of polar
filaments. Phys. Rev. Lett. . 92:078101.
Manville, J.-B., P. Bassereau, S. Ramaswamy, and J. Prost. 2001.
Active membrane aspirations studied by micropipette
aspirations. Phys. Rev. E. 64:021908.
Neves, S.R., P. Tsokas, A. Sarkar, E.A. Grace, P. Rangamani, S.M.
Taubenfeld, C.M. Alberini, J.C. Schaff, R.D. Blitzer, Moraru, II,
and R. Iyengar. 2008. Cell shape and negative links in
regulatory motifs together control spatial information flow in
signaling networks. Cell. 133:666-80.
Plowman, S.J., C. Muncke, R.G. Parton, and J.F. Hancock. 2005. H-
ras, K-ras, and inner plasma membrane raft proteins operate in

nanoclusters with differential dependence on the actin
cytoskeleton. Proc Natl Acad Sci U S A. 102:15500-5.
Ramaswamy, S., and M. Rao. 2007. Active-filament hydrodynamics:
instabilities, boundary conditions and rheology. New J. Phys. . 9
423.
Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes.
Nature. 387:569-72.
144
Publications
1. Nanoclusters of GPI-anchored proteins are formed by cortical

actin-driven activity.
Goswami D, Gowrishankar K, Bilgrami S, et al
Cell. 2008 Dec 12;135(6):1085-97.PMID: 19070578
2. Nanoscale organization of hedgehog is essential for long-

range signaling.
Vyas N, Goswami D, Manonmani A, Sharma P, Ranganath HA, et

al Cell. 2008 Jun 27;133(7):1214-27. PMID: 18585355
3. Precise positioning of myosin VI on endocytic vesicles in vivo.

Altman D*, Goswami D*, Hasson T, Spudich JA, Mayor S.
PLoS Biol. 2007 Aug;5(8):e210. PMID: 17683200
4. A DNA nanomachine that maps spatial and temporal pH

changes inside living cells.
Modi S, MGS, Goswami D, Gupta GD, Mayor S, Krishnan Y
Nat. Nanotech. 4, 325 - 330 (2009)
145
Synopsis
Thesis Title: Modes of nano-scale clustering of GPI-

anchored protein at the cell surface.
Name: Debanjan Goswami
Degree: Doctor of Philosophy

Subject: Cell Biology
Submitted to: Tata Institute of Fundamental Research

(Deemed University)
Thesis Supervisor: Prof. Satyajit Mayor
Institute Address: National Centre for Biological Sciences,

Tata Institute of Fundamental Research,
UAS- GKVK Campus, Bellary Road,
Bangalore. 560065. India.
Date of Submission: 10-June-2008
Signature of Supervisor Signature of Candidate
146
Synopsis
Introduction
A hallmark in the understanding of cell membrane organization

and structure was encapsulated in the Fluid Mosaic model (Singer and
Nicolson, 1972), where the membrane was visualized as an
equilibrated two-dimensional fluid – a passive mixture of proteins
dissolved in a sea of lipids. According to this model, all lipids and
proteins (ratio varies from 1:4 to 4:1) diffuse freely at all length-scale on
the surface of the cell (Frye and Edidin, 1970). Over the last decade,
the concept of a compartmentalized membrane has emerged where
the cell surface is not a homogeneous mixture, but is segregated into

domains. The mechanism of formation and maintenance of such
domains is hypothesized as arising due to the interaction between
specific lipids such as cholesterol and sphingolipids and associated
proteins. Compartmentalized regions or domains on cell membrane are
referred as ‘lipid-rafts’. ‘Lipid-rafts’ are proposed to be involved in a
variety of important biological roles including endocytosis, trafficking,
signaling complex formation (Simons and Ikonen, 1997). Although
numerous biological functions have been ascribed to ‘lipid-rafts’, the
mechanism behind their formation, structure and dynamics remain
highly debated (Mayor and Rao, 2004).
Phase segregated domains have been demonstrated in artificial

membranes composed of ternary mixtures of lipids. These mixtures
exhibit three distinct phases depending on temperature and
composition: gel (so), liquid ordered (lo) and liquid disordered (ld).
Above the chain melting temperature (Tm), the hydrocarbon chains of
lipids are floppy, disordered and loosely packed. This is known as
liquid (disordered) phase (ld). The ld phase has short range positional
correlation. Below Tm, lipids with saturated long acyl chains are tightly
147
Synopsis
packed and form a phase called the ‘gel phase’. The hydrocarbon
chains are oriented and ordered. The positional correlations in the
plane of the bilayer are long range. However, below Tm, in the
presence of cholesterol, long saturated acyl-chains remain oriented but
the positional correlations are short range, like in a liquid. This is known
as liquid ordered phase (lo). The diffusion coefficient of lipids in lo
phases are higher than in ‘gel-phase’, but lower than in the ld phase.
Since the rigid cholesterol molecule is inserted inside the lipid
molecules (in gel phase), the surface area per lipid molecule in the lo
phase is larger than in gel phase. However, above Tm in the presence
of cholesterol, no macroscopic phase segregation was observed. But
by spectroscopic studies, such as nuclear magnetic resonance (NMR)
and electron spin resonance (ESR), the two fluid state (lo and ld) was
shown to exist together (Sankaram and Thompson, 1990; Vist and
Davis, 1990). Below Tm, whereas the gel phase is generally observed,
in presence of high cholesterol (>20 mol%) gel phase is replaced by lo
phase and the two fluid (liquid) phases (lo and ld) can coexist (Brown
and London, 2000). Experimentally, when the ternary mixture was
brought down to below Tm of specific lipid species, these molecules
form liquid ordered phases (lo phase) in coexistence with disordered
phases (ld). These lo phases coalesce into large scale domains which
are resolvable by optical microscopy. It is this lo phase that is thought to
be relevant and analogous to ‘lipid-rafts’ in biological systems.
Since, the lo domains exist as large-scale phase-segregated

domains, it was expected that they could be observed in biological
membranes using techniques such as fluorescence microscopy,
electron microscopy, optical tweezers, single molecules studies and
biochemical treatments (chemical cross-linking). However, in contrast
to the situation in artificial membrane, none of the above techniques
have been able to detect the presence of any large scale lo domain on
148
Synopsis
the native cell membrane (Mayor and Rao, 2004; Munro, 2003). In this
scenario, several investigators tested the interaction of detergents with
membrane. This technique has been used to assess the ‘fluidity’ of
biological membrane (Helenius and Simons, 1975). It was argued that
if biological membrane contains ‘gel-like’ lo patches, similar to artificial
membrane, these would be insoluble in cold-nonionic detergents (for
example Triton X-100). Consequently, it has been shown that when cell
membranes are extracted with cold (4°C) Triton X-100, a non-ionic
detergent, a small fraction of insoluble membrane residue consisting of
specific subsets of lipids and proteins – called ‘detergent resistant
membrane’ (DRM), is observed (Simons and Ikonen, 1997).
Compositionally, DRM has been correlated to the lo domain on the
model membrane (London and Brown, 2000). This membrane fraction

is enriched in cholesterol, sphingomyelin and many lipid-tethered
proteins such as non-receptor tyrosine kinase,
glycosylphosphatidylinositol anchored proteins (GPI-APs), etc (Simons
and Ikonen, 1997). However, careful biophysical studies in artificial
membrane have showed that Triton X-100 (TX-100) can induce
formation of ‘more ordered phase’ in model membranes from native
‘disordered’ phase (Heerklotz, 2002; Heerklotz et al., 2003).
Furthermore, it has been observed that TX-100 extracted (4°C) DRM
composition matches with the ‘lo’ domain obtained in the ternary phase
diagram at 37°C, but not with the composition of the lo phase at 4°C
(de Almeida et al., 2003). That means detergent extraction can also
change the composition of preexisting domain on any artificial
membrane. So, a priori existence of lo domain or ‘lipid-raft’ on native
cell membrane and its composition remains questionable (Mayor and
Rao, 2004).
The scale of lipid domains in cell membrane has remained

controversial; in a few experimental attempts, the size of lipid raft has
149
Synopsis
been estimated from <10nm to 700nm (Anderson and Jacobson,
2002). Various biophysical tools such as Förster’s Resonance Energy
Transfer (FRET), chemical cross linking, single particle tracking,
fluorescence correlation spectroscopy, laser trap have been used to
investigate the size of large scale organization of different molecules
(lipids, GPI-APs, toxins, trans-membrane proteins) at the cell surface.
However, all these measurements failed to provide consensus scale of
the preexisting lipid domains at the cell surface.
GPI-APs have served as marker for rafts since they associate

with DRMs and form nanoclusters in a cholesterol sensitive fashion.
The nanoclusters were characterized by measuring homo-FRET
between fluorescently labeled GPI-APs on the native cell membrane. In

contrast to hetero-FRET where FRET is monitored between two
different fluorophore, in the homo-FRET process, energy transfer
between two like fluorophores mey be measured if they are in close
proximity (<10nm distance) (Varma and Mayor, 1998). This non-
invasive technique showed presence of sub-resolution (<70nm)
clusters of GPI-APs at the live cell surface (Varma and Mayor, 1998).
These sub-resolution clusters are sensitive to cholesterol and
sphingolipid content in the membrane. However, lack of measurable
hetero-FRET between donor and acceptor fluorescent labeled GPI-APs
on the cell surface contradicted the possibility of nanoclusters in sub-
resolution domain (Kenworthy and Edidin, 1998). A resolution of this
controversy was obtained when Sharma et al measured the scale of
GPI-AP organization. They theoretically modeled thegradual change in
homo-FRET efficiency observed upon photobleaching of fluorophore-
labeled GPI-APs and it to obtain the size of GPI-AP structures giving
rise to the FRET signals (Sharma et al., 2004). Moreover, Sharma et al
provided an explanation for lack of detectable hetero-FRET between
donor-acceptor pair labeled GPI-AP molecules in nanoclusters by
150
Synopsis
calculating the theoretical values of average hetero-FRET between
them (Sharma et al., 2004).
Properties of GPI-AP organization till date can be summarized

into following points: a) GPI-APs remain in a small fraction of
nanoclusters, 20-40% of the total protein on the cell surface, consists
of 2-4 molecules per cluster. b) nanoclusters are cholesterol sensitive.
c) Nanoclusters are always maintained a particular concentration
irrespective of surface protein concentration. This feature does not
obey the ‘law of mass action’ – represents a non-equilibrium state. d)
Multiple GPI-AP can cohabit within a cluster. This means clusters are
not frozen and they do exchange molecules with monomers present at
the cell surface.
This description provides an average picture of GPI-AP

organization at the cell surface at 20°C which lacks information about
the dynamics of the aggregation process or its spatial heterogeneity; it
also lacks explanation for non-equilibrium state of the whole process.
The present status and understanding of properties of ‘lipid-rafts’

in the view of GPI-AP organization at the cell surface evokes two
obvious questions. First, about the steady state dynamics and
distribution of nanocluster on the cell surface and second, about the
mechanism of maintenance of such unusual organization. Therefore, I
decided to address the following set of questions and attempt to
answer them by developing necessary tools:
1. The diffusivity of nanocluster vs. monomers at the cell

surface.
2. How the nanoclusters are distributed on the cell surface,
which requires high resolution anisotropy imaging.
151
Synopsis
3. The steady state dynamic properties of nanocluster on the
cell surface – the formation and fragmentation process.
4. Mechanism of maintenance of nanoclusters.
5. Possibility of inducing alteration in the organization.
Results
New experimental tools
Before going into the results, I would like to introduce two new
experimental strategies that I have developed to address these
questions.
1) I have developed a novel fluorescence intensity-anisotropy

trace (FIAT) assay. Here, I measure the intensity and the anisotropy
from a confocal volume on cell surface illuminated with a multi-photon
laser couple to a single photon counting device. In this assay, I locally
perturb the density of fluorophore-labeled folate receptor (FR-GPI) at
the surface of CHO cells by multi-photon (MP) confocal excitation
starting at time t=0 upto t=t1 which I specify as the first illumination
time; then the laser is switched off for a waiting time, tw; after this a
second illumination was perused for a time, t2. I follow the dynamical
response in anisotropy and fluorescence intensity simultaneously from
the same volume, where intensity represents the local concentration of
the protein and the value of the anisotropy reports on the
oligomerization status of these receptors.
2) I used a custom designed line-scanning microscope with high

spatio-temporal resolution for time-lapse anisotropy of emission
fluorescence imaging. A region (1μm2) was chosen on a fluorophore
labeled cell for bleaching and 80% fluorophore was bleached within
152
Synopsis
that area using high-intensity pulse from a separate bleaching laser.
Following the bleaching of fluorophore-FR-GPI at the centre of the
illuminated area, I follow the recovery of intensity and anisotropy in this
area at discrete time points. I name this assay as anisotropy recovery
after photobleaching (ARAP).
3) A new application of real time fluorescence dynamics has

been implemented to measure homo- and hetero-FRET on the surface
of a living cell. Using a pulsed laser (80MHz) and a time correlated
single photon counting device, I measure fluorescence lifetime of donor
fluorescence (in case of estimating hetero-FRET) and time resolved
anisotropy of fluorophore tagged GPI molecules (in case estimating
homo-FRET).
Detection of hetero-FRET by fluorescence lifetime

measurements
Using labeled acceptor and donor pairs, fluorescence lifetime

measurements report significant changes in donor lifetimes consistent
with the study GPI-AP organization reported in Sharma et al (Sharma
et al., 2004). I have used analogues of folic acid (a ligand for folate
receptor, FR-GPI), conjugated to two different fluorophores having
significant overlap in excitation and emission spectra (an important
criterion for FRET). Cells, expressing FR-GPI, labeled with increasing
acceptor to donor ratio shows gradual increase in energy transfer
efficiency. Energy transfer efficiencies at increasing acceptor to donor
ratio shows similar profile as predicted theoretically in Sharma et. al.
(Sharma et al., 2004). Thus, these fluorescence lifetime measurements
have the sensitivity to detect nanoclusters organization using FRET.
153
Synopsis
Aerolysin toxin alters organization of GPI-AP
Aerolysin is a plasma membrane pore-forming toxin, which, after

activation by carboxy-terminal cleavage, binds GPI-anchored proteins
in the membrane (Fivaz et al., 2002) and alters the oligomerization
status of the lipid anchored protein by heptamerizing (Tsitrin et al.,
2002). This heptamer eventually forms an aqueous pore in the
membrane. I have explored the alteration of GPI-AP nanocluster during
the process of toxin-induced GPI-AP clustering at the cell surface. Both
using homo- and hetero-FRET measurements, I show that the tools I
have established are able to describe the altered oligomerization status
of GPI-AP clusters.
GPI-AP nanoclusters are immobile and distributed

heterogeneo usly at the surface of living cells.
To study the diffusivity of GPI-AP clusters and monomersat the

cell surface I observed fluorescent tagged GPI-AP under high
resolution (where lateral and axial resolution being 260nm and 890nm
respectively) anisotropy imaging setup via line scanning confocal
microscope. Confocal anisotropy images show two distinct type of
optically resolvable regions on the cell surface – a. low average
fluorescence anisotropy regions, associated with flat regions of the cell
membrane; b. regions with high average anisotropy, associated with
membrane overlying protrusive actin architecture such as dynamic cell
edges, such as membrane ruffles or leading edges of lamellipodia. In
an ARAP experiment, I bleach the fluorophore tagged GPI-AP within a
region of 1μm2 in the flat part of a cell. I find that while the fluorescence
intensity recovers substantially, the anisotropy in the bleached spot
does not recover at 20°C. However, in the regions surrounding the
bleached areas, the anisotropy remains unchanged during the assay.
Interestingly, at 37°C, while the fluorescence intensity recovers rapidly
after bleaching, the original depolarized anisotropy value is eventually
154
Synopsis
recover, after a long delay. This suggests that nanoclusters are
immobile and can only be reformed at physiological temperature after a
certain time interval.
Steady state dynamics of GPI-AP nanoclusters
In a FIAT experiment, at 20°C, the anisotropy in the illuminated

volume starts out at a depolarized value which represents a mixture of
nanoclusters and monomers. Then as intensity goes down rapidly (an
effect of photobleaching), and reaches a steady state where
photobleaching and lateral diffusion compensates each other. The
fluorescence anisotropy shows a sharp initial rise (corresponding to
rapid loss of homoFRET). and saturates at a high value, which is a

characteristic of isolated monomers in the membrane (A∞). On the
other hand, at 37°C, the anisotropy rises during t1 and saturates to a
value significantly lower than A∞. During tw, recovery of intensity
indicates fluorophores diffuse in from the surrounding regions. After the
recovery time, the fluorescence anisotropy at 20°C, begins with the
same saturation value obtained at the end of the first illumination. It
never recovers to the depolarized value, obtained in the beginning of t1.
In contrast, at 37°C, there is an almost complete restoration of the
original depolarized anisotropy value after tw, implying that there is
substantial reassembly of nanoclusters from monomers at 37°C.
In collaboration with Kripa Growrishankar, I have tried to fit the

FIAT data precisely using a theoretical model to extract aggregation-
fragmentation kinetics of nanoclusters. The changes in fluorescence
intensity and anisotropy from the fluorophores present in the confocal
volume can be modeled by reaction-diffusion type equations,
incorporating diffusion of monomers and nanoclusters (diffusion
coefficients, D1 and Dc), bleaching of fluorophores (bleach rate, b), and
the interconversion between monomers and nanoclusters (aggregation
155
Synopsis
and fragmentation rates, ka and kf). The intensity and anisotropy
profiles can be obtained again knowing the monomer and nanocluster
anisotropy, Am and Ac.
Using the model, we determined the temperature (15°C to 37°C)

dependent aggregation-fragmentation kinetics. A heterogeneous
distribution of formation kinetics was observed for each temperature on
the cell surface. We determined the typical rates at each temperature
and tried to describe the process by a characteristic way of
representing temperature dependent chemical kinetics ─ an Arrhenius
plot. The Arrhenius plot of intercoversion shows two distinctly different
slopes with a crossover at 24°C. Above 24°C the slope is almost zero
and is steep below 24°C, suggesting a non-Arrhenius type behavior.
Membrane composition is important for formation of

nanoclusters
Cells, when treated with mβCD (methyl β-cyclodextrin) which

extracts cholesterol from the cell membrane, affects reformation of
clusters as detected in FIAT and ARAP assays at 37°C; whereas
intensity recovered anisotropy did not recovered in both experiments. It
has been shown previously, that GPI-AP nanoclusters are sensitive to
levels of cholesterol in the plasma membrane. It turns out that
cholesterol plays a key role for nanocluster formation and
maintenance.
Role of cortical actin in nanocluster dynamics
I have perturbed actin polymerization using Jasplakinolide (Jas)

or Latrunculin (Lat) at 37°C to explore the involvement of actin for
maintenance of nanoclusters at the membrane. Application of Jas or
Lat during the waiting period of FIAT assay prevents the restoration of
the starting depolarized anisotropy value, although the fluorescence
156
Synopsis
intensity recovers completely. Similar results were obtained when actin
polymerization was perturbed before the experiment in both ARAP and
FIAT assay. This clearly shows that cortical actin is actively involved in
interconversion dynamics of GPI-AP monomers and clusters.
Conclusions
In my thesis, I have shown following results:
1. I have estimated energy transfer efficiency in GPI-AP

organization by hetero-FRET method with varying labeling
ratios of acceptor and donor fluorescent probe. The profile

of the energy transfer efficiency is similar to the theoretical
prediction.
2. I have also shown that both homo- and hetero-FRET
measurement are complementary technique to estimate
altered oligomerization status of GPI-APs obtained upon
generating larger scale oligomer. In general, these
techniques together can be reliably used to determine scale
of molecular organization at the nanometer scale.
3. On the cell surface nanoclusters are immobile at different
length scale (from confocal area to 1μm2), but monomers
remain mobile.
4. Nanoclusters of GPI-AP are formed at physiological
temperature from existing monomers and maintained at a
specific concentration irrespective of the amount of the
protein present at plasma membrane. Therefore, this
system remains unequilibrated.
5. I measure the rate of formation and fragmentation of
nanoclusters with FIAT assay at multiple temperatures. In
collaboration Kripa G. and Dr. Madan Rao, we show the
157
Synopsis
inteconversion rate between monomer and clusters do not
exhibit Arrhenius type behavior.
6. Finally, I show that the formation of nanoclusters is
sensitive to the membrane composition (levels of
cholesterol) and cortical actin activity.
Future direction
It is still unclear what other factors could be involved in this

dynamic process. In addition the molecular links between the CA and
the exoplasmic lipid-tethered protein is poorly understood
158
Synopsis
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Modes of nano-scale clustering of GPI-anchored

protein at the cell surface.

for the degree of

National Centre for Biological Sciences

This thesis is a presentation of my original research work.

The work was done under the guidance of Professor Satyajit

Fundamental Research, Bangalore, India.

In my capacity as supervisor of the candidate’s thesis, I certify

Prof. Satyajit Mayor

I have tried to consolidate in this section the reminiscence from my

During my graduate studies, my thesis supervisor, Jitu, contributed

guidance. He has always given me the liberty to do experiments of my choice and

My thesis committee members Dr. Madan Rao, Dr. Mathew K. Mathew

Sameera Bilgrami, the person from whom I learned cell culturing,

I found the desired approach towards the interdisciplinary field of science

Entering undergrad college and choosing Microbiology honours course

University. It did modify the orientation and foundation of my understanding of

In my high school, Jadavpur Vidyapith, a first grade Bengali medium

My parents, the reason for my existence and whose contribution cannot

trusted my independent decisions and they have confidence in me. My sister, a

TABLE OF CONTENTS ......................................................................................X

1.B.i: Structure of plasma membrane ............................................................... 2

MATERIALS AND METHODS ................................................................................... 19

3.A: INTRODUCTION ............................................................................................... 30

3.C: LINE-SCANNING CONFOCAL MICROSCOPE .............................................................. 46

CHAPTER 6 ........................................................................................................... 109

CORTICAL ACTIN DRIVEN STEADY STATE DYNAMICS OF GPI-AP MONOMERS AND

cell surface irrespective of temperature. ....................................................... 113

CHAPTER 7 ........................................................................................................... 141

PUBLICATIONS ..................................................................................................... 145

SYNOPSIS ............................................................................................................. 146

A hallmark in the understanding of cell membrane organization

1.B.i: Structure of plasma membrane

The plasma membrane is assembled from a variety of lipids

common membrane lipids have this amphipathic character in common,

1.B.ii: The GPI-anchored proteins and its discovery

Amongst several peripheral membrane proteins,

1.B.iii: The structure and biosynthesis of GPI-APs

Following their discovery, extensive research commenced to

(ER). The GPI transamidase is a membrane-bound multi subunit

The GPI anchor is synthesized with an unsaturated fatty acyl

1.B.iv: The membrane association of GPI-APs

A well popular notion about GPI-APs is its putative association

such as sphingolipids and cholesterol, termed ‘rafts’. As shown

1.C: State of the membrane – ‘Raft Hypothesis’

1.C.i: Artificial membrane – description of ‘rafts’

Phase segregated domains have been demonstrated in artificial

1.C.ii: Rafts in biological membrane redefined

Since, the l o domains exist as large-scale phase-segregated

membrane. This technique was used to assess the ‘fluidity’ of

1.D: Biophysical methods

The scale of lipid domains in cell membrane has remained

been estimated from <10nm to 700nm (Anderson and Jacobson,

GPI-APs have served as marker for rafts since they associate

In contrast to hetero-FRET where FRET is monitored between

donor-acceptor pair labeled GPI-AP molecules in nanoclusters by

There are groups of researchers who have also shown slowing

Previous studies in our laboratory, using fluorescence

Properties of GPI-AP organization till date can be summarized

This description provides an average picture of GPI-AP

1.E: Objective of the thesis

The present status and understanding of properties of ‘lipid-rafts’