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THESIS for DOCTORAL DEGREE (Ph.D.) From THE DEPARTMENT OF MICROBIOLOGY, TUMOR AND CELL BIOLOGY Karolinska Institutet, Stockholm, Sweden THE ATYPICAL RHO GTPASE RHOD AND ITS ROLE IN CELLULAR DYNAMICS Magdalena Blom Stockholm 2017 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-print AB 2017 © Magdalena Blom, 2017 ISBN 978-91-7676-684-2 The atypical Rho GTPase RhoD and its role in cellular dynamics THESIS FOR DOCTORAL DEGREE (Ph.D.) By Magdalena Blom Principal Supervisor: Opponent: Professor Pontus Aspenström Professor Theresia Stradal Karolinska Institutet Helmholtz Centre for Infection Research, Department of Microbiology, Tumor and Cell Braunschweig Biology (MTC) Examination Board: Co-supervisor(s): Professor Ann-Kristin Östlund Farrants Dr Annica Gad Stockholm University Karolinska Institutet Department of Molecular Biosciences, Department of Medical Biochemistry and The Wenner-Gren Institute Biophysics (MBB) Associate Professor Ingela Parmryd Dr Katarina Reis Uppsala University Karolinska Institutet Department of Medical Cell Biology Department of Microbiology, Tumor and Cell Biology (MTC) Associate Professor Jonas Fuxe Karolinska Institutet Department of Microbiology, Tumor and Cell Biology (MTC) ABSTRACT Despite its discovery 20 years ago, the accumulated knowledge about the Rho member RhoD is scarce. Instead, the vast majority of studies concerning Rho GTPases has focused on the three classical members RhoA, Cdc42 and Rac1. In contrast, RhoD is considered an atypical Rho GTPase, with an aberrant GTP/GDP cycling and an unknown regulation. Like most Rho GTPases, altered RhoD protein levels result in actin cytoskeleton reorganization. We found that increased RhoD protein levels lead to a less dynamic actin cytoskeleton, while RhoD silencing leads to more pronounced actin-containing structures, such as stress fibers, cortical actin and ruffles, depending on cell type. Actin-dependent processes, such as cell migration and cell proliferation, are significantly affected in absence of RhoD in fibroblasts. Moreover, we have shown that endogenous RhoD, as well as its interaction partner WHAMM, localize to the Golgi apparatus. Silencing or overexpression of RhoD or WHAMM leads to a dispersion of the Golgi apparatus, suggesting a role of these proteins in Golgi homeostasis. In addition, protein transport from the ER to the plasma membrane is delayed both when overexpressing and silencing RhoD and WHAMM, as measured by the VSV-G protein transport assay. One of the first described functions of RhoD was its regulatory role in endosome fusion and trafficking. We found that the localization to vesicles is independent of the nucleotide-bound status of RhoD. However, only the GTP-bound RhoD can localize to the plasma membrane. In contrast, RhoD must be inactivated for fusion of RhoD positive vesicles. Deleting the unique N-terminal of RhoD leads to an altered distribution and characteristics of RhoD positive vesicles. Taken together, this thesis elucidates the role of RhoD in three different dynamic cellular processes; reorganization of the actin cytoskeleton, Golgi homeostasis and vesicle transport and fusion. LIST OF SCIENTIFIC PAPERS I. M. Blom, K. Reis, V. Nehru, H. Blom, A.K.B. Gad, P. Aspenstrom RhoD is a Golgi component with a role in anterograde protein transport from the ER to the plasma membrane. Exp Cell Res 2015 May 1;333(2):208-219 II. M. Blom, K. Reis, J. Heldin, J. Kreuger, P. Aspenstrom The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration. Exp Cell Res 2017 Mar 15;352(2):255-264 III. M. Blom, K. Reis, P. Aspenstrom RhoD localization and function are dependent on the GDP/GTP-bound state and unique N-terminal motif. Manuscript (2017) CONTENTS I. INTRODUCTION 1. Rho GTPases ........................................................................................................ 1 1.1 The Rho GTPase family .......................................................................... 1 1.2 Regulation of Rho GTPases .................................................................... 4 1.3 Rho effector proteins ................................................................................ 7 1.4 The Rho GTPase RhoD ........................................................................... 8 2. Actin ..................................................................................................................... 9 2.1 Polymerization and depolymerization ..................................................... 9 2.2 Actin structures ...................................................................................... 12 2.3 Actin myosin contraction ....................................................................... 14 2.4 Focal adhesions ...................................................................................... 14 2.5 RhoA, Cdc42, Rac1 downstream pathways .......................................... 16 2.6 Actin-driven cellular processes ............................................................. 18 2.7 RhoD and actin ....................................................................................... 19 3. The Golgi apparatus ........................................................................................... 20 3.1 Structure of the Golgi apparatus ............................................................ 20 3.2 Functions of the Golgi apparatus ........................................................... 21 3.3 Golgi homeostasis .................................................................................. 21 3.4 Actin at the Golgi apparatus .................................................................. 22 3.5 RhoD and the Golgi apparatus............................................................... 22 4. Vesicle dynamics ............................................................................................... 23 4.1 Early, late and recycling endosomes ..................................................... 23 4.2 Vesicle trafficking .................................................................................. 24 4.3 RhoD and vesicle dynamics .................................................................. 24 II. AIMS OF THE THESIS .......................................................................................... 27 III. RESULTS AND DISCUSSION .............................................................................. 29 Paper I..................................................................................................................... 29 Paper II ................................................................................................................... 31 Paper III .................................................................................................................. 34 IV. ACKNOWLEDGEMENTS ..................................................................................... 37 V. REFERENCES ......................................................................................................... 39 LIST OF ABBREVIATIONS ADF actin-depolymerization factor ADP adenosine diphosphate Arp2/3 complex actin related proteins 2/3 complex ATP adenosine triphosphate Cdc42 cell division control protein 42 homolog CP capping protein CRIB cdc42/Rac-interactive binding motif Dbl diffuse B cell lymphoma DH Dbl-homology DHR Dock Homology Region DRF Diaphanous-related formin ECM extracellular matrix EEA1 early endosome antigen 1 Ena/VASP enabled/vasodilator-stimulated phosphoprotein ER endoplasmic reticulum FA focal adhesion F-actin filamentous actin FH domain formin homology G2 gap phase 2 GA Golgi apparatus G-actin globular actin GalT 1,4-galactosyltransferease GAP GTPase activating protein GDI guanosine nucleotide dissociation inhibitor GDP guanosine diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GM130 Golgi matrix protein 130 GMF glial maturation factor GRASP65/55 Golgi reassembly-stacking protein of 65/55 kDa GTP guanosine triphosphate IRSp53 insulin-receptor substrate p53 kDa kilo Dalton LIMK Lin11, Isl-1, Mec-3 domain kinase mDia mammalian diaphanous protein MLC myosin light chain MLCP myosin light chain phosphatase NPF nucleation promoting factor N-WASP neuronal Wiskott-Aldrich syndrome protein PDE phosphodiesterase PDGF platelet derived growth factor PH pleckstrin homology Rab Ras-like proteins in brain Rac Ras-related C3 botulinium toxin substrate Ras Rat sarcoma RBD Rho binding domain REM Rho effector homology Rho Ras homolog Rnd Round ROCK Rho associated protein kinase TC10 Teratocarcinoma 10 TGN46 trans-Golgi network 46 t-SNARE target-soluble N-ethylmaleimide sensitive factor attachment protein receptor VCA domain verprolin, cofilin, acidic domain VSV-G vesicular stomatitis virus protein G WAVE WASP family-verprolin homologous protein WHAMM WASP homolog-associated protein with actin, membranes and microtubules wt wild-type I. INTRODUCTION This thesis aims to present data to elucidate the role of RhoD as a regulator of dynamic cellular processes. Being a member of the Rho GTPases, it is not surprising that RhoD exert a regulatory role on the actin cytoskeleton. However, this thesis also presents data to illuminate RhoD in its role as a regulator of vesicle dynamics and Golgi homeostasis. 1. Rho GTPases 1.1 The Rho GTPase family Nature has developed a variety of ways to regulate the activity of cellular proteins. One common strategy is phosphorylation, often leading to activation of a protein. Other examples are
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