the distinct roles of cytohesin 2 and cytohesin 3 in cell adhesion and migration

109 Pages·2011·2.4 MB·English

by  Seung Ja Oh

The Pennsylvania State University

The Graduate School

Department of Biochemistry and Molecular Biology

THE DISTINCT ROLES OF CYTOHESIN 2 AND
CYTOHESIN 3 IN CELL ADHESION AND MIGRATION

A Dissertation in

Biochemistry, Microbiology and Molecular Biology

by

Seung Ja Oh

© 2011 Seung Ja Oh

Submitted in Partial Fulfilment
of the Requirements
for the Degree of

Doctor of Philosophy

August 2011
The dissertation of Seung Ja Oh was reviewed and approved* by the following:

Lorraine C. Santy
Assistant Professor of Biochemistry and Molecular Biology
Dissertation Advisor
Chair of Committee

Wendy Hanna-Rose
Associate Professor of Biochemistry and Molecular Biology

Melissa Rolls
Assistant Professor of Biochemistry and Molecular Biology

Graham Thomas
Associate Professor of Biochemistry and Molecular Biology

Okhee Han
Assistant Professor of Nutritional Sciences

Scott B. Selleck
Professor of Biochemistry and Molecular Biology
Head of the Department of Biochemistry and Molecular Biology

*Signatures are on file in the Graduate School

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ABSTRACT

In tissues, epithelial cells are normally stationary; however, the cells become migratory in
cases of developmental morphogenesis, wound healing, or metastasis. Cellular migration is a
well-coordinated process that requires altered cell shape and polarity. Recent studies showed that
these processes involve small GTPases of the ADP-ribosylation Factor (ARF) family. In
particular, ARF6 regulates endocytosis and recycling of junction proteins, as well as an actin
cytoskeleton assembly during cell migration. A large number of GEFs, which likely regulate
ARFs at different locations and during different processes, activate ARF6. However, not all of
these GEFs‟ functions are defined. This study investigates the roles of cytohesin ARF-GEFs in
cell adhesion and migration. First, the study shows that cytohesin 2/ARNO and cytohesin
3/GRP1 have distinct functions for cell adhesion, spreading, and migration. Cytohesin 2/ ARNO
expression enhances cell adhesion, spreading, and migration while cytohesin 3/GRP1 expression
reduces cell adhesion, spreading, and migration. Second, the study also shows that cytohesin
2/ARNO is required for β1 integrin recycling while cytohesin 3/GRP1 is dispensable for this
process. Third, the study finds that cytohesin 2/ARNO and cytohesin 3/GRP1 have differential
localizations in spreading cells. Cytohesin 2/ARNO localizes at both the periphery and interior
regions, while cytohesin 3/GRP1 exclusively locates at the plasma membrane. Since the findings
indicate that the closely related cytohesin 2/ARNO and cytohesin 3/GRP1 have differential
functions and localizations, the investigation also considers the sequence differences of cytohesin
2/ARNO and cytohesin 3/GRP1 that produce the differential effects. Finally, this research
demonstrates that the number of glycine residues (2G or 3G) in the Pleckstrin Homology (PH)
domain of cytohesins is the critical difference underlying opposing effects of cytohesin 2/ARNO
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and cytohesin 3/GRP1 on cell adhesion, spreading and migration. The glycine residues in the PH
domain are a known site for PIP2 and PIP3 bindings, and the PH domain of cytohesin 2/ARNO
and cytohesin 3/GRP1 have different binding affinity to PIP2 and PIP3. Based on all data, this
study proposes that ARNO and GRP1 recruitment to different membrane domains by their
different selectivities for PIP2 and PIP3 is responsible for their differential effects on cell
adhesion, spreading, and migration. This is the first demonstration of the unique roles of
cytohesin 2/ARNO and cytohesin 3/GRP1.

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TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………………………......ix
ABBREVIATIONS…………………………………………………………………………...…xi
CELL LINES…………………………………………………………………………...….…..xiii
ACKNOWLEDGMENTS……………………………………………………………………..xiv
CHAPTER 1: Introduction……………………………………………………………………….1
Epithelial Cells…………………………………………………………………………….2
Cell Migration……………………...……………………………………….……………..3
Various types of cell migration……………..…………………………….……………….4
Integrin……………..……………………………………………………….……………..5
Cancer and Metastasis………...…………………………………………………………...7
ADP-ribosylation Factor 6 (ARF6) …………...…………………………………….……9
Cytohesin Family……………...………………………………………………………....11
Aims of Study…………………..……………………………………………….….……14
Hypothesis………………………………………….………………………….…………14
Specific Aim 1…………...………………………………………………………………15
Specific Aim 2………………...…………………………………………………………15
Specific Aim 3……………...………………………………………………...………….16

CHAPTER 2: Materials and Methods………………………………………………….……….17
Cells and Reagents…………………………………………………………………….…18
siRNA and Plasmids……………………………………………………………………..18
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Primer Seqeunces………………………………………………………...………………19
Adhesion Assay………………………………………………………………………….21
Analyzing β1 integrin recycling by Microscopy………………………………………...21
Biochemical β1 integrin recycling Assay…………………………………….………….23
Surface β1 integrin Labeling Assay………………………………………………….…..23
Migration Assay……………………………………………………………………….…23
Spreading Assay…………………………………………………….……………………25
Flow Cytometry ……………...………………………………………………………….25
RT-PCR………………………………………………………………………………….26
Deconvolution Microscopy………………………………………………………...…….26

CHAPTER 3: Differential Effects of Cytohesin 2 and 3 on Cell Adhesion, Spreading and
Migration…………………………………………..…………………………….27

Rationale…………………………………………………….…………………………...28
Cytohesins are critical for cell adhesion to fibronectin……………………………...…..29
ARNO and GRP1knockdown induce differential effects on cell spreading…………….33
ARNO and GRP1 knockdown have opposing effects on cell migration………………..33

CHAPTER 4: Determination of Guanine-nucleotide Exchange Factor (GEF) which regulates
β1 integrin recycling………………………………….………………………….36

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Rationale…………………………………………………………………………………37
Cell surface β1 integrin………………………………………….……………………….38
β1 integrin recycling requires cytohesins………………………………………………..41
Only ARNO is required for β1 integrin recycling……………………………………….44
Localization of cytohesins in spreading cells……………………………………………44

CHAPTER 5: Identification of the cytohesin 2/ARNO and cytohesin 3/GRP1 sequence
differences that underlie involvement in integrin recycling……………..….…...48

Rationale…………………………………………………………………………………49
Cytohesin 2/ARNO and cytohesin 3/GRP1 Hybrid Constructs…………………………49
Cell adhesions of domain swapped ARNO and GRP1………………………….….……52
ARNO (3G) vs. GRP1 (2G) ……………………………………………………….…….54
Localization of 2G ARNO and 3G GRP1 in spreading cells…………………….………56

CHAPTER 6: Discussion………………...……………………………………………………..62

Differential functions of cytohesin 2/ARNO and cytohesin 3/GRP1 in cell adhesion,
spreading and migration………………………………………………………………….64
Differential localizations of cytohesin 2/ARNO and cytohesin 3/GRP1………..……….64
Opposing functions of ARNO and GRP1 on cell adhesion and spreading induced by PH
domain……………………………………………………………………………………65
β1 integrin recycling pathway ……………………………………………………...……66
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Integrin Traffic…………………………………………………………………...67
Counteracting function of GRP1…………………………………………..…….71

CHAPTER 7: Future direction………………………………………………………………….75

Rescue of integrin recycling ……………………………………………………………76
Experimental approach……………………………………………….………….76
Investigation of co-localization of cytohesin 2/ARNO and cytohesin 3/GRP1 with PIP2
and PIP3…………………………………………………………………………………78
Experimental approach……………………………………………………….….79
E-cadherin recycling study………………………………………………………………80
Experimental approach…………………………………………………………..81

REFERENCES………………………………………………………………………………….84

APPENDIX A: siRNA knockdown efficiency………………………………………………….92

APPENDIX B: Source of contents……………………………….…………………..…………93

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LIST OF FIGURES

Figure 1.1 The metastatic process………………………………………………………….…….8
Figure 1.2 The exchange cycle of GTPases…………………………………………...………..10
Figure 1.3 The Sec7 Family of ARF GEFs……………………………………………………..13
Figure 2.1 Surface Antibody Binding Recycling Assay…………………………………...…...22
Figure 2.2 Schematic of cell migration measurement…………………………………………..24
Figure 3.1 Cytohesin function is required for cell adhesion…………………………………....30
Figure 3.2 ARNO expression enhances cell adhesion to fibronectin, while cell adhesion is
repressed by GRP1 expression……………………………………………………………………………….31
Figure 3.3 ARNO knockdown reduces cell adhesion, while GRP1 knockdown enhances cell
Adhesion……………………………………………………………………………………………………………….32
Figure 3.4 Effect of cytohesin knockdown on the spreading of MCF-7 cells…………………………..34
Figure 3.5 ARNO and GRP1 knockdown have opposing effects on cell migration……………..….35
Figure 4.1 Cell surface expression of β1 integrin in cells with altered levels of cytohesin
Expression…………………………………………………..………………………………………………………..39
Figure 4.2 Cell surface levels of β1 integrin in Hela cells overexpressing GFP, GFP + ARNO,
or GFP + GRP1 measured by flow cytometry…………………………………………………….….40
Figure 4.3 β1 integrin recycling is detected by the surface antibody binding in MCF-7 cells....42
Figure 4.4 β1 integrin recycling requires cytohesins. (A) β1 integrin is retained intracellularly
in cells treated with SecinH3………………………………………………………………………………..43
Figure 4.5 β1 integrin recycling requires ARNO……………………………………………………………………45
Figure 4.6 Differential localization of cytohesin 2 and 3 in spreading cells…………………………….46
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Figure 4.7 Differential localization of cytohesin 2 and 3 in the Z-dimension………………….….…..47
Figure 5.1 Constructs of domain swapped cytohesin 2/ ARNO and cytohesin 3/ GRP1….….…..51
Figure 5.2 ARNO with GRP1 PHPB expression does not enhance cell adhesion…………….……..53
Figure 5.3 Constructs of domain swapped ARNO and GRP1…………………………………………………54
Figure 5.4 ARNO with GRP1 PH expression reduces cell adhesion……………………..………………..56
Figure 5.5 2G ARNO expression reduces cell adhesion to fibronectin while wild type ARNO
(3G) expression enhances cell adhesion………………………..……………………………………….58
Figure 5.6 Effect of 2G ARNO or 3G GRP1 expression on the spreading of MCF-7 cells………59
Figure 5.7 Similar localization of 2G ARNO and wild type GRP1 in spreading cells………..……60
Figure 5.8 Differential localization of 3G GRP1 and wild type GRP1 in spreading cells……..…61
Figure 6.1 Model for β1 integrin recycling……………………………………………….…... 69
Figure 6.2 ARF6 signaling pathways…………………………………………………….….…72
Figure 6.3 Model for counteracting function of GRP1……………………………………...…73
Figure 7.1 E-cadherin Recycling was detected by the surface biotin labeling technique and the
Calcium-Switch Assay………………………………………………………………82
Figure 7.2 E-cadherin recycling requires cytohesins………………………………………………………….…..83
Figure A-1 Transfection of siRNA reduces expression of cytohesin 2/ARNO and cytohesin
3/Grp1………………………………………………………………………………92

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