University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2010 The Role of the Exocyst in Exocytosis and Cell Migration Jianglan Liu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons Recommended Citation Liu, Jianglan, "The Role of the Exocyst in Exocytosis and Cell Migration" (2010). Publicly Accessible Penn Dissertations. 201. https://repository.upenn.edu/edissertations/201 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/201 For more information, please contact [email protected]. The Role of the Exocyst in Exocytosis and Cell Migration Abstract The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells expressing constitutively active c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of Exo70 blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly regulating Arp2/ 3-mediated actin polymerization. Using pyrene actin assay and total internal reflection fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the function of the exocyst in exocytosis and cell migration. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biology First Advisor Wei Guo Keywords Exocytosis, Exocyst, Invadopodia, The Arp2/3 complex, cell migration, actin polymerization Subject Categories Cell Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/201 THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION JIANGLAN LIU A DISSERTATION in BIOLOGY Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 Supervisor of Dissertation _________________________________ Wei Guo, Associate Professor of Biology Graduate Group Chairperson _________________________________ Paul Sniegowski, Associate Professor of Biology Dissertation Committee Tatyana Svitkina, Associate Professor of Biology Michael Lampson, Assistant Professor of Biology Kimberly Gallagher, Assistant Professor of Biology Margaret Chou, Associate Professor of Pathology and Laboratory Medicine Mickey Marks, Associate Professor of Pathology and Laboratory Medicine ACKNOWLEDGEMENTS First of all, I would like to give my deepest gratitude to my mentor Dr. Wei Guo. I regard Wei as my scientific father as he guided me into the wonderful scientific field and directed me all the way till now. I am so impressed with his enthusiasms and curiosity on science, his persistence on research and his extensive knowledge. And I really appreciate that he gives me inspiring advices and warm encouragements in all the time of research. I would benefit from his mentoring throughout my entire scientific career. Secondly, I really appreciate all the members in my thesis committee: Prof. Tatyana Svitkina, Prof. Margaret M. Chou, Prof. Mickey Marks, Prof. Kimberly Gallagher and Prof. Michael A.Lampson. They have committed much time and efforts to follow my research and have always been a great source of constructive suggestions and encouragements. I enjoy the wonderful time that I have spent with all the previous and current members of Guo lab. They make the lab such a pleasant place for everyday work. They are not only my scientific partners, but also my incredible friends. In particular, I would like to thank Dr. Bing He who has offered me so much help and valuable advices. I appreciate Dr. Xiaofeng Zuo for his contribution to the first part of this study. I am grateful to Dr Xiaoyu Zhang and Jian Zhang for their nice help since I ii entered the lab. I also want to thank Dr. Kelly Orlando for her help on my scientific English writing. Finally, I want to thank all other lab folks for their constant discussions and encouragements. I would also like to extend thanks to the people who have been instrumental in completion of this work. I am grateful to Prof. Susette C. Mueller, Dr. Vira V. Artym and Mr. Peter Johnson for their collaboration on the invadopodia research; Prof. Yale Goldman and Dr. Yujie Sun for their collaboration on the TIRFM experiments; Dr. Jian Jing for giving me advices for zymography experiments; Prof. Roberto Dominguez, Dr. Grzegorz Rebowski and Dr. Boczkowska Malgorzata for their help on the pyrene actin assay. Finally, I would like to give my special thanks to my family. I want to thank my parents with my heart for their love, support and encouragement throughout my life. Most of all, I would give my thanks to my dear husband, Peng Yue. He is my labmate, my soulmate and my lifemate. His solid help for my research and his endless love for my life make me strong enough to go this far. I want to give my last but most special thanks to my little angel Shannon Xianning Yue. You make mommy’s life complete. Love you forever. iii ABSTRACT THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Jianglan Liu Supervisor: Dr. Wei Guo The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells iv expressing constitutively active c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of Exo70 blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly regulating Arp2/3-mediated actin polymerization. Using pyrene actin assay and total internal reflection fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the function of the exocyst in exocytosis and cell migration. v Table of Contents Chapter 1. Introduction································································································· 1 1.1 Overview··············································································································· 1 1.2 The exocyst complex···························································································· 3 1.3 Phosphoinositides in cell regulation and membrane dynamics·························· 56 1.4 Cell invasion and invadopodia structures··························································· 60 1.5 Thesis overview··································································································