TOG PROTEINS ARE SPATIALLY REGULATED BY RAC-GSK3β TO CONTROL INTERPHASE MICROTUBULE DYNAMICS. Kathryn P. Trogden
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1 TOG PROTEINS ARE SPATIALLY REGULATED BY RAC-GSK3β TO CONTROL INTERPHASE MICROTUBULE DYNAMICS Kathryn P. Trogden A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology in the College of Arts and Sciences. Chapel Hill 2015 Approved by: Stephen Rogers Victoria Bautch Stephanie Gupton Benjamin Major Kevin Slep
2 2015 Kathryn P. Trogden ALL RIGHTS RESERVED ii
3 ABSTRACT Kathryn P. Trogden: TOG Proteins are spatially regulated by Rac-GSK3β to control interphase microtubule dynamics (Under the direction of Stephen L. Rogers) Within a cell, the ends of individual microtubules switch between three different phases: growth, shrinkage and pause without affecting the total mass of microtubule polymer. This inherent property of microtubules is termed dynamic instability. During interphase, microtubule dynamics are regulated by a class of proteins that interact primarily with the plus end of the microtubule (+TIPs). How these proteins are regulated, either through interactions with each other or through signaling pathways in the cell are poorly understood. Two +TIP protein families, the XMAP215 family, known as Mini spindles (Msps) in Drosophila and the CLASP family, known as Orbit, contain arrays of tubulin binding TOG domains. We show that in Drosophila S2 cells Orbit is phosphorylated by the kinase GSK3β, similar to what has been reported for mammalian CLASPs. At the periphery of the cell, GSK3β is inhibited by the Rho GTPase Rac and Orbit is no longer phosphorylated. Orbit is then able to interact with Msps through their C-termini, allowing Msps to bind to the lattice. Msps lattice binding is important for proper microtubule dynamics as mutations that disrupt this interaction lead to aberrant dynamics. This interaction requires the scaffolding protein Sentin to bring the two proteins into contact at the periphery of the cell. Further work has shown a role for another Rac effector kinase, PAK, in this pathway. This interaction may also be important for migration in Drosophila cells. Depletion of +TIPs leads to changes in cell movement, indicating that microtubule dynamics may play a role in motility. iii
4 ACKNOWLEDGEMENTS I would like to acknowledge my advisor, Steve Rogers, and my committee, Vicki Bautch, Stephanie Gupton, Ben Major, and Kevin Slep, for their creative input and encouragement throughout my dissertation progress. I would also like to thank the members of the Rogers lab, past and present. A special thanks for Josh Currie and Derek Applewhite for continual help and dicussions about my project. Also to Kim Peters for not sugar coating things and providing realistic views on graduate school. A special thanks to the people who made graduate school survivable, Lauren Slevin, Tom Lane, Fatima Manzoor, Jennifer Heppert, Diana Chong and Joy Meserve. Finally, I would like to thank my parents, Jeff and Anita Trogden, for doing my taxes, supporting, if not understanding my choice to stay in school and not get a real job and for making an effort, if not always succeeding, in saying Drosophila. iv
5 PREFACE Chapter 2 is a manuscript in review at MBoC. I performed all of the experiments and anaylsis and drafted the figures. Stephen Rogers and I designed experiments and drafted the manuscript. Trogden, K.P., and Rogers, S.L. (2015). Spatial regulation of Drosophila Mini-spindles by Rac and GSK3β requires its interaction with Orbit. Mol Biol Cell. In review. Chapter 3 describes an ongoing project that is a collaborative effort between Stephen Rogers and me to further explore the pathway described in Chapter 2. I performed all of the experiments and anaylsis. Chapter 4 describes an ongoing project to study the role of the pathway decribed in Chapter 2 in cell migration. All expirements and anaylsis were performed by me. Experimental design was overseen by Derek Applewhite and Steve Rogers. v
6 TABLE OF CONTENTS LIST OF TABLES... xiii LIST OF FIGURES... xiv LIST OF ABBREVIATIONS AND SYMBOLS... xvi CHAPTER 1 : INTRODUCTION... 1 Summary... 1 Microtubules are inherently dynamic... 1 Dynamic Instability is regulated in cells by Microtubule Associated Proteins... 4 End Binding (EB) Proteins... 5 SLAIN/Sentin Proteins... 9 Cytoplasmic Linker Associated Proteins (CLASPs) Dis1/XMAP215 Proteins Kinesin-13/KinI Depolymerase Motor Proteins The Role of +TIPs in Cell Migration References CHAPTER 2 : SPATIAL REGULATION OF DROSOPHILA MINI-SPINDLES BY RAC AND GSK3Β REQUIRES ITS INTERACTION WITH ORBIT Summary Introduction Results There are two populations of microtubules in Drosophila S2 cells Rac and GSK3β regulate Orbit and Msps localization in S2 cells vii
7 Msps is regulated by GSK3β through Orbit Sentin is necessary for Msps and Orbit to interact The C-termini of Msps and Orbit interact Msps mutants that cannot bind Orbit increase microtubule growth Discussion Experimental Procedues Cell Culture and RNA interference Immunofluorescnece and live cell imaging Immunoprecipitation/Western-blotting Molecular biology and transfection Microtubule and actin retrograde flow measurements References Tables Supplemental Figures CHAPTER 3 : PLK4 PB3 IS A DIVERGENT PB DOMAIN WITH SPECIES- DEPENDENT ROLES AND A VARIABLE STRUCTURE Summary Introduction Results Rac1 regulates Orbit and Mini-spinldes lattice binding, but Mtl does not Orbit and Mini-spindles lattice binding is regulated downstream of Rac by the PAK kinases Discussion Experimental Procedues Cell-Culture and RNA interference Immunofluorescence and live-cell imaging viii
8 Molecular biology and transfection Colocalization analysis References CHAPTER 4 : DROSOPHILA D25 CELLS ARE MOTILE AND REQUIRE PROPER MICROTUBULE DYNAMICS FOR NORMAL CELL MOVEMENT Summary Introduction Results D25 cell have lamellipodia and move in a non-directional manner Pioneer microtubules grow into the lamellipodia at the leading edge of D25 cells Proper microtubule dynamics are important for regular cell migration of D25 cells Msps and Orbit lattice association is regulated by Rac1 in D25 cells Disscussion Experimental Procedures Cell Culture and RNA interference Immunofluorscence and live cell imaging Molecular biology and transfection Measurement of Cell Movement References CHAPTER 5 : CONCLUSIONS AND FUTURE WORK TIPs interact at the plus end in a hierarchical, regulated manner TIPs play an important role in cell migration TIPs in Drosophila cell migration Microtubules and +TIPs play a role in Drosophila development Concluding remarks References ix
9 LIST OF TABLES Table 2.1. Microtubule dynamics parameters x
10 LIST OF FIGURES Figure 1.1. Microtubules stochastically switch between three states Figure 1.2. Domain Structure of +TIP Families Figure 1.3. Pathways Downstream of Rac at the Leading Edge of Motile Mammalian Cells Figure 2.1 Orbit and Msps localization is regulated by Rac-GSK3β signaling Figure 2.2. Orbit is phosphorylated by GSK3β in the linker region between TOG2 and TOG Figure 2.3. Orbit and GSK3β levels regulate Msps recruitment to the microtubule lattice Figure 2.4. Sentin expression is required for the Orbit-Msps interaction, but is not regulated by Rac Figure 2.5 Msps interacts with Orbit Figure 2.6 Measurement of microtubule dynamics Figure 2.7. Model Figure 2.S1. Endogenous Orbit and Msps are regulated by Rac-GSK3β signaling Figure 2.S2. Orbit is phosphorylated by GSK3β in the linker region between TOG2 and TOG Figure 2.S3. EB1 is not regulated by Rac and Sentin can interact with Orbit Supplemental Figure 2.S4. Msps interacts with Orbit Figure 2.S5. Rates of actin retrograde flow is altered only by changes in Rac activity Figure 3.1 Rac1, and not Mtl, regulates Orbit and Msps lattice association Figure 3.2 PAK1 and PAK3 regulate the lattice association of Orbit and Msps redundantly Figure 4.1 D25 cells are able to move in a non-directional manner Figure 4.2 Microtubules are important for D25 cell movement xi
11 Figure 4.3 Msps and Orbit localize to microtubules in D25 cells and respond to CA-Rac Figure 5.1 Orbit and KLP10A can co-immunoprecipitate xii
12 LIST OF ABBREVIATIONS AND SYMBOLS CH ConA CA CuSO4 CLASP CLIP EB FBS GFP GEF MAP MTOC +TIP Mtl Msps PAK PBS RNAi TIRF μm calponin homology concanavalin A constitutively active copper sulfate Cytoplasmic linker associated protein Cytoplasmic linker protein End binding fetal bovine serum green fluorescent protein guanine nucleotide exchange factor microtubule associated protein microtubule organizing center microtubule plus end interacting protein Mig2-like protein Mini spindles p21 activated kinase phosphate-buffered saline RNA interference total internal reflection fluorescence micrometer xiii
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