The regulation of integrin-mediated cell adhesion and spreading by the actin-binding protein ...

169 Pages·2010·6.31 MB·English

The regulation of integrin-mediated cell adhesion and
spreading by the actin-binding protein filamin A

By

Hugh Kim

A thesis submitted in conformity with the
requirements for the degree of

Doctor of Philosophy

Graduate Department of Dentistry

University of Toronto

© Copyright by Hugh Kim 2010
ABSTRACT

Title: The regulation of integrin-mediated cell adhesion and spreading by the actin-binding
protein filamin A
Degree: Doctor of Philosophy (Ph.D.)
Year of convocation: 2010
Graduate Department of Dentistry
University of Toronto

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton,
matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants
of these interactions in early events in cell spreading are not defined. I found that the actin-
binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed
extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short
hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of
collagen binding sites on β1 integrin reduced (p<0.05) cell spreading and the localization of
filamin A at cell extensions. Knockdown of filamin A reduced β1 integrin occupancy by
collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin
A and β1 integrin. Based on mass spectrometry screening of potential filamin A interacting
proteins I examined the interaction of filamin A with the intermediate filament protein
vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and
exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast,
knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface β1
integrin expression and β1 integrin activation. Knockdown of filamin A reduced vimentin
phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of
filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell
spreading, vimentin phosphorylation and β1 integrin surface expression and activation were
all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell
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spreading was also reduced by siRNA knockdown of protein kinase Cε. By
immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found
an association between filamin A and vimentin. Filamin A also associated with protein
kinase Cε, which was enriched in cell extensions. In vitro pull-down assays using deletional
mutants of purified filamin A showed that both vimentin and protein kinase Cε bound to a
region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with
full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin
phosphorylation and the cell surface expression of β1 integrins. I conclude that interactions
of filamin A with vimentin and protein kinase Cє may be important for the trafficking and
activation of β1 integrins and cell spreading on collagen.
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ACKNOWLEDGEMENTS

First and foremost, I would like to thank Chris McCulloch for his superb mentorship and
supervision. His infinite generosity, wisdom and incredible talents as a clinician-scientist have
provided me with a solid foundation on which to build my career. I shall always be grateful to
Chris for his support during my Ph.D. studies and sincerely hope that my own contributions to
science and the dental profession will justify his efforts.

I would also like to thank Mike Glogauer, Boris Hinz and the late Jaro Sodek for serving on my
Ph.D. advisory committee and for many helpful discussions. I am grateful to Michal Opas for
allowing me to participate in his laboratory’s studies and to Richard Ellen for his tireless
dedication to reseach training. I thank Don Brunette and Babak Chehroudi (The University of
British Columbia) for encouraging my early interest in research as well as Ed Putnins for his
friendship and advice. I am also grateful to René Voyer (Université de Montréal) and Ira Sy for
their mentorship during the early stages of my dental career.

My day-to-day existence in the laboratory was extremely stimulating, challenging and fun.
There are many people I need to thank for this. Carol Laschinger and Cheung Lo generously and
patiently provided excellent tutelage in immunoblotting and cell culture, respectively. I thank
my good friend Wilson Lee for his invaluable flow cytometric analyses, his good humor and for
keeping the lab organized and efficient. I was fortunate to share numerous professional and
scientific exchanges (but most importantly, many jokes) with Matthew Chan, Amy Yeung, Reza
Termei, Mindy Pho, Ibrahim Mohammad, Dhaarmini Rajshankar, Jan Kuiper, Anne Koehler
and Roland Leung. Their collegiality and willingness to make time for coffee or beer made my
stay at the University of Toronto a special one.

Special thanks to Fumihiko Nakamura (Harvard Medical School) for his friendship and for
generously providing filamin reagents. Thanks to Yulia Shifrin, who provided the filamin-
knockdown cells that were essential for my experiments. Thanks are due to Elena Bajenova for
introducing me to confocal microscopy, Marilyn Murphy for her administrative assistance,
Claire Hong for her excellent plasmid preparations and TIRF micrographs and Pam Arora for
performing the actin assembly assay (Fig. 3-3a). Thanks also to Bob Chernecky and Jian Wang
for providing SEM micrographs (Fig. 2-1).

I thank my wife Christine (the self-declared “lab widow”) for her patience and keen sense of
humor. I also thank my in-laws, Graham and Loretta Lee, for their kindness and generosity. I am
forever indebted to my parents for their guidance, support and for the values they instilled in me.
During the past 3½ years, I was particularly fortunate to catch up (and again, share many jokes)
with my sister Suh, my brothers Hyung and Hoe, as well as their families.

I gratefully acknowledge the fellowship support provided by the Canadian Arthritis Network
(2006-2008), the University of Toronto Harron Fund (2006-2008), the CIHR Cell Signals
program (2006-2008) and the Heart and Stroke Foundation of Canada (2008-2010).
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TABLE OF CONTENTS

Abstract ii
Acknowledgements iv
Table of contents v
Preface viii
List of tables x
List of figures xi

Chapter 1: Literature Review 1
Overview 2
Introduction: cell adhesion and spreading 3
1. Filamin structure, isoforms and expression 4
a. General structure 4
b. Isoforms 8
c. Regulation of filamin expression 9
d. Structural aspects of filamin binding 10
2. Filamin A: role in cell adhesion, spreading and migration 13
a. Filamin A and the intracellular traffic of cell surface molecules 14
b. Filamin A as a regulator of integrin function 15
c. Filamin A, small GTPases and actin assembly 17
3. Filamin: an integrator of cytoskeletal systems? 18
a. Filamin A and microtubules 19
b. Filamin A and the intermediate filament cytoskeleton 19
c. Filamin, vimentin and integrin-mediated cell adhesion/spreading 21
4. Filamin A participates in protein kinase-mediated cell signaling 24
Concluding remarks 25

Chapter 2: Filamin A regulates cell spreading and survival via β1 integrins 26
Abstract 27
Introduction 28
Materials and Methods 30
Reagents 30
Preparation of substrates 30
Cell culture and filamin A knockdown 30
Scanning electron microscopy 31
Immunoblotting of plasma membrane proteins 31
Immunoprecipitation 32
Immunohistochemistry 32
TUNEL staining 33
Intracellular calcium 33
Statistical analysis 33
Results 35
Filamin A localizes to cell extensions of spreading human gingival fibroblasts 35
Surface chemistry and surface topography affect cell spreading and filamin A
distribution 37
Filamin A regulates cell spreading on smooth collagen surfaces 37
Filamin A and cell spreading on non-integrin binding substrates 42
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Filamin A is required for adhesion-dependent survival 44
Filamin A and [Ca2+] are independent regulators of cell spreading 44
Filamin A regulates cell spreading via β1 integrins 46
Discussion 53
Filamin A is required for cell spreading 53
Filamin A and intracellular calcium 55
Filamin A is a determinant for β1 integrin-ligand binding 56
Filamin A promotes cell survival 57

Chapter 3: Filamin A is required for vimentin-mediated cell adhesion and
spreading 58
Abstract 59
Introduction 60
Materials and Methods 62
Reagents 62
Cell culture 62
Immunoprecipitation 63
Isotope-coded affinity tag analysis 63
Small interfering RNA knockdown 63
Preparation of purified filamin A, dot-blot and pull-down assays 64
Immunoblotting of plasma membrane-associated proteins 65
Actin monomer incorporation 66
Rac and Cdc42 activation assay 66
Immunohistochemistry 66
Flow cytometry 67
Statistical analysis 67
Results 68
Filamin A associates with vimentin during cell spreading 68
Filamin A and vimentin coregulate cell spreading 69
Filamin A, but not vimentin, affects actin assembly in spreading cells 71
Filamin A and vimentin regulate β1 integrin expression and activation 73
Vimentin phosphorylation is required for β1 integrin expression, β1 integrin
activity and cell spreading 77
Filamin A binds vimentin directly in vitro and in spreading cells 80
Filamin A is a determinant of vimentin phosphorylation and reorganization 81
Discussion 91
Filamin A and vimentin coregulate cell spreading 91
Filamin A and vimentin coregulate β1 integrin expression and activation 93
Vimentin phosphorylation is required for β1 integrin activity and cell
spreading 93
Filamin A is required for PKC-mediated vimentin phosphorylation 94

Chapter 4: Regulation of cell adhesion to collagen via β1 integrins is dependent
on interactions of filamin A with vimentin and protein kinase C epsilon 96
Abstract 97
Introduction 98
Materials and Methods 100
Reagents 100
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Cell culture 100
siRNA knockdown 101
Immunoblotting of plasma membrane-associated proteins 101
Cell adhesion assay 102
Preparation of filamin A constructs 103
Pull-down assays 104
Immunohistochemistry and cell spreading assays 105
Flow cytometry 105
Statistical analysis 105
Results 107
Filamin A is required for vimentin re-organization and cell spreading 107
Filamin A regulates vimentin phosphorylation by protein kinase Cε 107
Cell surface expression of β1 integrins and cell adhesion strength 111
Filamin-vimentin and filamin-PKCε interactions 115
Cell spreading is regulated by direct filamin-vimentin and filamin-PKCε
Binding 117
Vimentin phosphorylation and cell surface β1 integrin 119
Discussion 126
Filamin A is required for vimentin phosphorylation and re-organization
during cell spreading 126
Filamin A, vimentin and PKCε co-regulate cell surface β1 integrin activation
and adhesion 127
Filamin A binds both vimentin and PKCε along repeats 1-8 127
Filamin A-vimentin and filamin A-PKCε binding is required for vimentin
phosphorylation, cell spreading and cell surface β1 integrin expression 128

Chapter 5: Conclusions and future directions 130
1. Summary and conclusions 131
2. Future directions 132

References 135

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PREFACE

This thesis is presented in a “publishable” format in which the experimental data of chapters 2, 3
and 4 have already been published. Chapter 1 (literature review) will be submitted for
publication as a review article and therefore includes descriptions of the experimental data from
chapters 2-4. Chapter 2 describes the role of filamin A in regulating the early phases of cell
spreading on collagen. Chapter 3 describes a novel interaction between filamin A and the
intermediate filament protein vimentin in the regulation of cell spreading. Chapter 4 describes
how filamin regulates the phosphorylation of vimentin by protein kinase C.

Publications from this thesis:

1. Kim H, Nakamura F, Lee W, Hong C, Pérez-Sala D, McCulloch CA. Regulation of cell
adhesion to collagen via β1 integrins is dependent on interactions of filamin A with
vimentin and protein kinase C epsilon. Exp. Cell Res., in press.
2. Kim H, Nakamura F, Lee W, Shifrin Y, Arora P, McCulloch CA. Filamin A is required
for vimentin-mediated cell adhesion and spreading. Am. J. Physiol. Cell Physiol.
2010;298(2):C221-36. Epub 2009 Sep 23.
3. Kim H, Sengupta A, Glogauer M, McCulloch CA. Filamin A regulates cell spreading
and survival via β1 integrins. Exp. Cell Res. 2008;314(4):834-836.

Other publications:

1. Papp S, Szabo E, Kim H, McCulloch CA, Opas M. Kinase-dependent adhesion to
fibronectin: regulation by calreticulin. Exp. Cell Res. 2008;314(6):1313-1326.
2. Papp S, Fadel M, Kim H, McCulloch CA, Opas M. Calreticulin affects fibronectin-
based cell-substratum adhesion via the regulation of c-Src activity. J. Biol. Chem.
2007;282(22):16585-16598.
3. Kim H, Murakami H, Chehroudi B, Textor M, Brunette DM. Effects of surface
topography on the connective tissue attachment to subcutaneous implants. Int. J. Oral
Maxillofac. Implants 2006;21(3):354-365.

Book chapters:

1. Kim H, McCulloch CA. Development of the periodontal ligament and alveolar bone. In:
Mao JJ, Nah HD (eds). Craniofacial Growth and Development, in press.
2. Hamilton DW, Ghrebi S, Kim H, Chehroudi B, Brunette DM. Surface topography and
cell behavior. In: Bowlin G, Wnek G (eds). The Encyclopedia of Biomaterials and
Biomedical Engineering (2005, pp. 1-11). New York, Dekker.

AWARDS:

Fellowship/salary support awards:

• 2008-2010: Research Fellowship, Heart and Stroke Foundation of Canada
• 2008: Canadian Institutes of Health Research (CIHR) Doctoral Research Award –
Frederick Banting and Charles Best Canada Graduate Scholarship (declined)
• 2008: Ontario Graduate Scholarship (declined)
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AWARDS, continued:

Fellowship/salary support awards, continued:

• 2006-2008: Postdoctoral Fellowship, Canadian Arthritis Network
• 2006-2008: CIHR Cell Signaling in Mucosal Inflammation and Pain Research Award
• 2006-2008: Harron Scholarship, University of Toronto

Research prizes and distinctions:

• 2008: Unilever-Hatton Award, First Place (Basic Science Category), International
Association for Dental Research (IADR)
• 2007-08: First place winner, Canadian Association for Dental Research-Institutes for
Musculoskeletal Health and Arthritis (CADR-IMHA) Research Competition
• 2008: Oral presentation award, Canadian Connective Tissue Conference (June 5-8,
2008)
• 2008: Poster presentation award (rank: # 1/80), CIHR-ICRH YI Forum (May 8-10,
2008)
• 2008: Travel Award, CIHR Institute of Circulatory and Respiratory Health YI Forum

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LIST OF TABLES

Table 1-1: List of common filamin- and vimentin-interacting partners 21

Table 3-1: List of filamin A-associated proteins identified by ICAT 68
x