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University of Cincinnati UNIVERSITY OF CINCINNATI Date: 1-Oct-2010 I, Jason Matthew Puglise , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Cell & Molecular Biology It is entitled: Roles of the Rac/Cdc42 effector proteins Pak and PIX in cytokinesis, ciliogenesis, and cyst formation in renal epithelial cells Student Signature: Jason Matthew Puglise This work and its defense approved by: Committee Chair: Robert Brackenbury, PhD Robert Brackenbury, PhD 11/1/2010 1,117 Roles of the Rac/Cdc42 effector proteins Pak and PIX in cytokinesis, ciliogenesis, and cyst formation in renal epithelial cells A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program of Cancer and Cell Biology of the College of Medicine by Jason M. Puglise M.Sc., Wright State University 2005 Committee Chair: Robert Brackenbury, Ph.D. ii ABSTRACT Puglise, Jason M. Ph.D., Cancer and Cell Biology Program. University of Cincinnati, 2010. Roles of the Rac/Cdc42 effector proteins Pak and PIX in cytokinesis, ciliogenesis, and cyst formation in renal epithelial cells. The p21-activated kinase 1 (Pak1) is a putative Rac/Cdc42 effector molecule and a multifunctional enzyme implicated in a wide range of cellular and biological activities. Although well-established as a regulator of cytoskeletal and microtubule dynamics, Pak1 influences centrosome behavior and plays a part in the cell cycle. We examine the role Pak1 and its binding partner Pak1-interacting exchange factor (PIX) play in centrosome dynamics and in cell cycle events in renal epithelial cells. Utilizing Madin-Darby canine kidney (MDCK) cells as our model system, we provide evidence that Pak1 and PIX function are important for proper centrosome functioning and cell division. We found that inhibition of the normal function of Pak or PIX leads to centrosome and cytokinesis defects. Moreover, our results reveal that Pak1 and PIX play a crucial role in renal epithelial morphogenesis and ciliogenesis, a function not previously described for Pak. Furthermore, our findings stress the importance of the Pak-PIX interaction in regulating cilia and acetylated tubulin dynamics, including lumen formation. Taken together, our work suggests that Paks are important for renal biology. iii COPYRIGHT JASON M. PUGLISE 2010 iv ACKNOWLEDGMENTS I want to thank various past and present members of the Zegers/ter Beest lab from the University of Cincinnati and from the University of Chicago for their contributions to my work. I want to thank my committee members: Dr. John Bissler, Dr. Robert Brackenbury (Committee Chair), and Dr. Wallace Ip for their time and cooperation. I want to express my gratitude to Dr. Karl Matlin, who played a role in my admittance and timely departure from the program. I’d like to sincerely thank my advisor, Dr. Mirjam Zegers, for her help and guidance on my dissertation. In addition, I’d like to thank my good friend, Steve Kioko, and my brother, Scott, for their advice and proofreading of my dissertation. Lastly, I’d like to dedicate this thesis to my family—particularly my father, stepmother, brother, sister, and step-uncle for their unwavering support and encouragement. Without their aid, I could not have overcome the obstacles nor endured the hardships that were imposed upon me. I am truly indebted to them for all their help, and am appreciative for having such a caring and understanding family. Truly, blood is thicker than water. “Our greatest glory is not in never failing, but in rising up every time we fail.” (Ralph Waldo Emerson) v TABLE OF CONTENTS Page INTRODUCTION.........................................................................................................................1 CHAPTER 1. General background and scope of dissertation I. Introduction to Rho GTPases and Pak family of kinases.........................................2 Background............................................................................................................2 Structure of Pak......................................................................................................6 Modes of action…………………………………………………………………..7 II. Paks as multifunctional kinases……………………………………………………..8 Regulation of cytoskeleton dynamics.....................................................................8 Role of Paks and small GTPases in cell cycle, cytokinesis, and spindle regulation……………………………….………………...…..……10 Role of Paks and small GTPases in microtubule dynamics.................................13 III. Centrosomes.............................................................................................................18 General.................................................................................................................18 Function………………………………………………………………………...19 Centrosome duplication........................................................................................20 Role of Paks and small GTPases in centrosome dynamics……………………..20 Consequences of centrosome malfunctioning and disease..................................21 IV. Cilia………………………………………………………………………………….23 General………………………………………………………………………….23 Structure………………………………………………………………………...23 Function………………………………………………………………………...24 vi TABLE OF CONTENTS (continued) Page Primary cilia as mechanosensors.........................................................................24 Primary cilia formation is linked to the centriole and cell cycle..........................25 Diseases associated with ciliary dysfunction……………………...……………27 V. Scope of dissertation................................................................................................27 CHAPTER 2. Pak and PIX regulate ciliogenesis and cytokinesis I. Introduction…………………………………………………………………………...33 II. Results………………………………………………………………………………..35 III. Discussion…………………………………………………………………………..47 CHAPTER 3. Pak and PIX control lumen formation I. Introduction…………………………………………………………………………...77 II. Results………………………………………………………………………………..82 III. Discussion…………………………………………………………………………..84 CHAPTER 4. Materials and methods I. Cell culture and cell lines……………………………………………………………..94 II. Western blotting (WB) and immunoprecipitation (IP)………………………………96 III. siRNA………………………………………………….…………...…………….…97 IV. Immunofluorescence (IF) confocal microscopy…………………………………….98 V. BrdU incorporation…………………………………………………………………100 vii TABLE OF CONTENTS (continued) Page CHAPTER 5. General discussion/ future goals & directions I. General discussion………………………………………………………………..…102 II. Future goals & directions……………………………………………………..…….107 REFERENCES...........................................................................................................................110 viii LIST OF FIGURES Page CHAPTER 1. Figure 1. Diagram of Rho GTPases (Rho, Rac, and CDC42) in their inactive and active states..................................................................................................................................29 Figure 2. Schematic diagram indicating features of Pak1 structure…………………………...30 Figure 3. General structure of cilia…………………………………………………………….31 Figure 4. The assembly and disassembly of primary cilia are coordinated with the centriole and cell cycle………………………………………………………………………….32 CHAPTER 2. Figure 1. Overexpression of dominant-negative Cdc42 promotes ciliogenesis……………….55 Figure 2. GIT1 and PAK localize to centrosome……………………………………………...58 Figure 3. Active Pak1 inhibits ciliogenesis over time…………………………………………60 Figure 4. Diminished ciliogenesis in active Pak1 mutant cells partially depends on Pak-PIX interaction……………………….…………………………………………….………62 Figure 5. Loss of βPIX leads to formation of Ac-tub rich cell extensions…………………….64 Figure 6. Ac-tub rich cell extensions induced by βPIX knockdown do not emanate from a basal body and associate with mitotic cells……………………………………………...66 Figure 7. Loss of Pak leads to formation of Ac-tub rich cell extensions……………………...68 Figure 8. Loss of PIX or Pak induces multinucleation and supernumerary centrioles………..70 Figure 9. Expression of kinase-dead Pak1 induces the formation of Ac-tub rich cell extensions which depends on Pak-PIX interaction………………………………………….......73 Figure 10. Expression of PIX mutant unable to bind to Pak induces Ac-tub rich cell extensions.....................................................................................................................................75 ix LIST OF FIGURES (continued) Page CHAPTER 3. Figure 1. Expression of active Pak1 induces formation of cyst-like lumens in two-dimensional MDCK culture……………………………………………………………......89 Figure 2. Formation of cyst-like lumens stimulated by Pak1-L107F depends on interaction with βPIX.....................................................................................................................................91 Figure 3. Expression of Pak1-L107F affects LN synthesis and secretion……………………..92 Figure 4. Accumulation of LN in lumens of 14-day old cultures of Pak1-L107F-expressing cells…………………………………………………………………...93 x INTRODUCTION The Rho GTPases Rho, Rac, and Cdc42 are pleiotropic regulators of many crucial cellular processes. As molecular switches, they interact and activate multiple downstream effectors to trigger signaling pathways. Pak kinases are putative effectors of Rac and Cdc42, and as multifunctional kinases, take part in a variety of biological activities. This dissertation focuses on the Rho GTPases and Paks and their
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