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Role of Rho-Family Guanosine Triphosphatase Effectors in Filopodia Dynamics

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Role of Rho-Family Guanosine Triphosphatase Effectors in Filopodia Dynamics ROLE OF RHO-FAMILY GUANOSINE TRIPHOSPHATASE EFFECTORS IN FILOPODIA DYNAMICS Arpan De A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2015 Committee: Carol Heckman, Advisor Paul Morris Daniel Wiegmann © 2015 Arpan De All Rights Reserved iii ABSTRACT Carol Heckman, Advisor Filopodia play sensory roles by acting like ‘antennae’ to sense the cell’s surroundings. In nerve growth cone, they promote motility towards an attractive cue or away from a repulsive one. Filopodia have been reported to be involved in wound healing, adhesion to the extracellular matrix and embryonic development. A number of cytoskeletal regulatory proteins have been implicated in regulating initiation, formation, maintenance and extension/retraction cycle of such protrusions. Some of these proteins either bind to the Rho family GTPases, Cdc42 and Rac, as effectors or function downstream of such effectors to regulate signaling pathways involved in cytoskeletal reorganization. The purpose of this study was to determine whether certain proteins, which had well- defined binding interfaces with proteins downstream of GTPase-regulated proteins, were implicated in filopodial dynamics. Synthetic binding peptides (BPs) were used to impede the interaction of Cdc42 and Rac with the effectors and to interfere at protein-protein binding interfaces downstream of the GTPases. Single cell peripheries were analyzed to determine the levels of positive outgrowths of filopodia. This assay allowed for rapid determination of whether the BPs had an effect on filopodia formation. BPs, namely IQGAP (132-140), ACK and Par6 showed a negative effect on filopodia, whereas IQGAP (84-93), PAK and WASP elevated filopodia formation or had no effect compared to control. When interactions of PAK2 with Abl and PAK4 with integrin-β5 were inhibited, filopodia prevalence increased. Based on several previous findings, the tyrosine kinase ACK is thought to mediate internalization, processing and trafficking pathways of cell surface receptors, especially iv epidermal growth factor receptors (EGFRs). ACK is an oncogene and its overexpression or mutation is implicated in several human cancers. During this study, when the E3 ubiquitin ligase, neural precursor cell expressed developmentally downregulated protein (Nedd4) was inhibited from binding to ACK, both cell periphery coverage with filopodia and number of cells showing filopodia, increased significantly compared to control. Since earlier findings have suggested that ACK and RTK (especially EGFR) are co-processed, this study suggests that, ACK-mediated RTK processing may play a role in regulating filopodial dynamics. v To my Advisor, Dr. Carol Heckman, my beloved parents, Mrs. Kankan De and Mr. Asim Kumar De and my beloved brother, Mr. Apratim De. vi ACKNOWLEDGMENTS I take the privilege to convey my sincerest and profound gratitude to Dr. Carol Heckman, whose valuable guidance, inspiration, love and support have immensely helped me to understand and learn the aspects of basic scientific research and gain insightful knowledge of the subject. I am thankful to Dr. Paul Morris, Dr. Daniel Wiegmann, Dr. Michael Geusz and Dr. Scott Rogers for constantly motivating me. I am indebted to Dr. Marilyn Cayer for her invaluable technical support and help during the study. I would like to thank Tania Biswas, Pratima Pandey and Francis Bugyei for their all along help. I am grateful to Pratima Pandey for sharing some data of her study and immensely helping me during the course of research. I convey my sincere thanks to Dr. Jeff Miner, Dr. Karen Root and the Department of Biological Sciences at Bowling Green State University for their continuous support. I have no words to express how much indebted I am to my beloved parents and family for believing in me and for their constant encouragement and unconditional love. I convey a very special note of thanks to my uncle, Mr. Arun Kumar De for being my invaluable support, philosopher and guide. Thank you, Ms. Ananya Banerjee for being my best friend and immensely helping me to stay strong, confident and focused to achieve my goal. I would like to thank all my friends for being there beside me and supporting me all along. I offer my heartfelt prayers to the Almighty for blessings. vii TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION ....................................................……………………… 1 1.1 Specializations implicated in cell motility.................................................... 1 1.2 Guanosine triphosphatase mediated regulation of protrusions........................ 5 1.2.1 Cell division cycle 42 (Cdc42)............................................................ 6 1.2.2 Ras related C3 botulinum toxin substrate (Rac).................................. 9 1.3 Working model of filopodia initiation and elongation .................................... 11 1.4 Adhesive proteins and regulatory kinases in filopodia.................................. 14 1.5 Proteins implicated in receptor trafficking..................................................... 15 1.5.1 ACK and its role in receptor trafficking................................................. 15 1.5.2 ACK has complex effects on receptor trafficking................................ 16 1.6 Eperimental model....................................................................................... ̀ 21 CHAPTER 2. MATERIALS AND METHODS ………………………. ............................ 24 2.1 Cell culture and chemicals ............................................................................. 24 2.2 Substrate Prepartion.............................................................................................. 25 2.3 Peptides designed to interrupt protein-protein interactions.................................. 26 2.4 Peptide delivery using BioPorter reagent.............................................................. 27 2.5 Analysis………………………………………………………….............……... 29 CHAPTER 3. RESULTS……………………………. ......................................................... 31 3.1 Assay for effect of binding peptides on filopodia dynamics.......................... 31 3.2 Peptides blocking downstream of GTPase effectors...................................... 35 3.3 Membrane traffaicing ̀ and filopodia............................................................... 43 viii CHAPTER 4. DISCUSSION…………………………………………………………… .... 47 4.1 Role of Cdc42 effectors and downstream proteins in filopodia dynamics...... 47 4.2 ACK plays a critical role in receptor trafficking............................................. 50 4.2.1 ACK binds constitutively and conditionally to other proteins............ 50 4.2.2 Overexpressed ACK inhibits receptor internalization by sequestering its binding partners......................................................................................... 52 4.2.3 Coordinate ACK and RTK trafficking and degradation............................ 54 4.2.4 Role of ACK mediated EGFR processing in filopodia dynamics............. 57 REFERENCES........................................................................................................................ 60 APPENDIX A. LIST OF ABBREVIATIONS……………………………………………. .. 72 ix LIST OF FIGURES Figure Page 1.1 Steps in filopodium initiation, elongation and retraction ........................................... 4 1.2 GTPases act as switch proteins .................................................................................. 5 1.3 Cdc42 pathway showing the different effectors and the cellular processes they generate ............................................................................................................ 8 1.4 Rac1 pathway showing the different effectors and the cellular processes they generate ............................................................................................................ 10 1.5 Formation of filopodia and lamellipodium showing the different proteins involved 12 1.6 The current working model for filopodia formation .................................................. 13 1.7 Graphical representation showing cyclical relationship predicted by the model for hypothetical predator and prey populations ............................................................... 22 2.1 Delivery of synthetic BPs into cells using BioPORTER reagent .............................. 28 2.2 Method of estimating coverage with filopodia .......................................................... 30 3.1 Percentage of cells showing filopodia after ACK-Nedd4 BP treatment versus control 38 3.2 Mean percent of cells showing filopodia after ACK-Nedd4 BP treatment in combination with other BPs ....................................................................................... 39 3.3 Percent coverage of cell periphery with filopodia after ACK-Nedd4 BP treatment versus control ………................................................................................................ 40 3.4 Mean percent coverage of cell periphery with filopodia after ACK-Nedd4 BP treatment in combination with other BPs.................................................................. 41 x 3.5 Percentage of cells showing filopodia after cAbl-PAK2 BP treatment versus control 42 3.6 Percentage coverage of periphery with filopodia after cAbl-PAK2 BP treatment versus control ............................................................................................................ 43 4.1 Schematic diagram showing ACK (1038 amino acid residues)
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