ROLE AND REGULATION OF JUXTANODIN IN ACTIN CYTOSKELETON OF OLIGODENDROCYTE MENG JUN NATIONAL UNIVERSITY OF SINGAPORE 2010 ROLE AND REGULATION OF JUXTANODIN IN ACTIN CYTOSKELETON OF OLIGODENDROCYTE MENG JUN (MBBS, MS, Peking University, Beijing, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS It is my great pleasure to present my deeply gratitude to the many people who made this thesis possible. First and foremost, I am extremely grateful to my Ph.D. supervisor, Associate Professor Liang Fengyi, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore. He provides good research direction, offers subjects and resources and penetrates criticism during my Ph.D. study. My sincere appreciation is to Professor Bay Boon Huat, Head of Department of Anatomy, for his constant help and encouragement. I am also very grateful to Professor Ling Eng Ang, the former Head of Department of Anatomy, for his continue encouragement and the opportunity to pursuer my Ph.D. study in NUS. My sincere acknowledgements are also devoted to those colleagues in our research group: Dr. Li Wenbo, Dr. Tran Manh Hung, Dr. Tang Junhong, Mr. Xia Wenhao, Ms. Wu Chun, Ms. Pooneh Memar Ardestani, Ms. Guo Jing and Ms. Wang Xie. The members in our group are warm-hearted and diligent. It is my great honor to work with them. I am also grateful to the administration and research officers in Department of Anatomy, for helping the department to run smoothly and for assisting me in many different ways. Mdm Ang Lye Gek Carolyne, Mdm Teo Li Ching Violet and Mdm Diljit Kour d/o Bachan Singh, Ms. Chan Yee Gek, Ms. Ng Geok Lan and Ms. Yong Eng Siang, deserve special mention. i Acknowledgements I would like to thank my friends in Singapore, especially Dr. Deng Yiyu, Dr. Jiang Boran and Dr. Feng Luo for helping me get through the difficult times, and for all the emotional support, entertainment, and caring they provided. Finally, I must always be grateful to my parents: they raise me and love me. I am deeply indebted to my wife’s love and patience; she supports our new family when I pursue my Ph.D. study. This thesis for PhD degree would be dedicated to her. ii Table of contents TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………….i TABLE OF CONTENTS ……………………………………………………… iii LIST OF TABLES AND FIGURES …………………………………………… .ix LIST OF ABBREVIATIONS ………………………………………………………xi LIST OF PUBLICATIONS ………………………………………………………xvii SUMMARY ………………………………………………………………………xviii CHAPTER INTRODUCTION……………………………………….1 1.1 Oligodendrocyte and its actin cytoskeleton……………………………………… 1.1.1 Oligodendrocyte……………………………………………………………….2 1.1.2 Actin cytoskeleton in oligodendrocyte……………………………………… .2 1.1.2.1 Cytoskeleton and its roles in oligodendrocyte…………………………….2 1.1.2.2 Actin-binding proteins in oligodendrocyte……………………………… .3 1.2 Actin cytoskeleton…………………………………………………………………5 1.2.1 Cytoskeletal elements………………………………………………………… 1.2.2 Actin……………………………………………………………………………7 1.2.3 Actin dynamics……………………………………………………………… 10 1.2.3.1 Nucleation……………………………………………………………… 11 1.2.3.2 Actin filament extension, disassembly and stabilization…………………13 1.2.4 Actin-binding proteins……………………………………………………… .14 1.2.4.1 Actin monomer binding proteins…………………………………………16 iii Table of contents 1.2.4.2 Actin filament capping proteins………………………………………….17 1.2.4.3 Actin filament severing proteins ……………………………………… .19 1.2.4.4 Actin filament bundling/crosslinking proteins………………………… .19 1.2.5 Actin-based cellular structures……………………………………………… 21 1.2.5.1 lamellipodia………………………………………………………………21 1.2.5.2 Filopodia………………………………………………………………….22 1.2.5.3 Stress fibers………………………………………………………………24 1.3 ERM proteins and Juxtanodin……………………………………………………26 1.3.1 ERM proteins…………………………………………………………………26 1.3.1.1 Structure of ERM proteins……………………………………………….26 1.3.1.2 Distribution and functions of ERM proteins…………………………… 28 1.3.1.3 Actin and membrane binding of ERM proteins………………………….29 1.3.1.4 Conformational regulation of ERM proteins…………………………… 30 1.3.1.5 Crosstalk between ERM proteins and Rho GTPases…………………….32 1.3.2 Juxtanodin…………………………………………………………………….36 1.3.2.1 Molecular character and expression of Juxtanodin………………………36 1.3.2.2 JN in myelination and specialization of the node of Ranvier……………38 1.4 The objectives of the current study………………………………………………39 CHAPTER MATERIALS AND METHODS…………………… .41 2.1 Chemicals……………………………………………………………………… .42 2.2 Oligonucleotides………………………………………………………………….42 iv Table of contents 2.3 Plasmids………………………………………………………………………….43 2.4 Cell lines………………………………………………………………………….44 2.5 Molecular biology……………………………………………………………… 46 2.5.1 Polymerase chain reaction…………………………………………………….46 2.5.2 Restriction and electrophoretic separation of DNA on agarose gels………….47 2.5.3 Ligation of DNA…………………………………………………………… .48 2.5.4 Transformation……………………………………………………………… 49 2.5.5 DNA plasmid preparation…………………………………………………….49 2.5.6 DNA sequencing…………………………………………………………… .49 2.5.7 Site-directed mutagenesis…………………………………………………… 50 2.5.8 Western blot………………………………………………………………… 51 2.6 Biochemical methods…………………………………………………………….53 2.6.1 Expression and purification of GST-tagged fusion proteins………………….53 2.6.2 Expression and purification of polyhistidine-tagged fusion proteins…………55 2.6.3 Purification of GST and GST-JN protein by gel filtration FPLC………………56 2.6.4 Buffer-exchange by cut-off centrifugal filters……………………………… .56 2.6.5 BCA protein assay…………………………………………………………….57 2.6.6 Actin filament co-sedimentation assay……………………………………… 57 2.6.7 F-actin bundling assay……………………………………………………… .60 2.6.7 Fluorescent measurement of actin assembly………………………………….60 2.7 Cellular biology………………………………………………………………… 61 2.7.1 Cultivation of cells……………………………………………………………61 v Table of contents 2.7.2 Transfection………………………………………………………………… .62 2.7.2.1 Electroporation of OLN-93 and CHO cells………………………………62 2.7.2.2 Chemical transfection of Cos1 cells with Lipofectamine……………… 62 2.7.3 Treatment of OLN-93 cells with Latrunculin A………………………………63 2.7.4 Immunocytochemistry (ICC)…………………………………………………64 2.7.5 Wound healing assay………………………………………………………….66 2.8 Statistical analysis……………………………………………………………… 68 CHAPTER RESULTS………………………………………………69 3.1 The role of Juxtanodin in actin dynamics……………………………………… 70 3.1.1 Juxtanodin specially bound F-actin………………………………………… 70 3.1.2 Juxtanodin interacted with F-actin through the last C-terminal 14 amino acid residues………………………………………………………………………71 3.1.3 Juxtanodin exhibited isoform preference of actin binding………………… .74 3.1.4 Juxtanodin had no activity of bundling or cross-linking………………… 76 3.1.5 Juxtanodin could not promote actin polymerization…………………………77 3.1.6 Juxtanodin inhibited F-actin disassembly in vitro……………………………79 3.1.7 Juxtanodin increased F-actin content of OLN-93 cells………………………81 3.1.8 Juxtanodin inhibited F-actin disassembly induced by Latrunculin A……… 84 3.2 The effect of Juxtanodin on actin-based cellular structures and behaviors………85 3.2.1 Juxtanodin promoted cellular aborization of OLN-93 cells………………….85 3.2.2 Juxtanodin promoted cell spreading of OLN-93 cells……………………… 87 vi Table of contents 3.2.3 Juxtanodin induced the formation of actin fibers of OLN-93 cells and localized along the side of actin fibers………………………………………88 3.2.4 Juxtanodin inhibited cell mobility by its C-terminal F-actin binding domain.90 3.3 The possible regulation of Juxtanodin by phosphorylation and RhoA GTPase………………………………………………………………………….93 3.3.1 Phosphorylation of T258 of Juxtanodin did not alter its influence on cellular morphology and actin cytoskeleton of OLN-93 cells………………………93 3.3.2 Phosphorylation of S278 of Juxtanodin abolished its influence on cellular morphology of OLN-93 cells……………………………………………….95 3.3.3 Phosphorylation of S278 of Juxtanodin abolished its influence on cellular actin cytoskeleton of OLN-93 cells……………………………………………….97 3.3.4 Inhibition of RhoA magnified the arborization of CHO cells induced by Juxtanodin………………………………………………………………… 99 CHAPTER DISCUSSION…………………………………………101 4.1 Binding of Juxtanodin with F-actin…………………………………………… 102 4.2 Effect of Juxtanodin on actin dynamics……………………………………… .103 4.3 The possible roles of JN in oligodendrocyte differentiation, migration, myelination and specialization of the node of Ranvier……………………… 105 4.4 Significance of regulation of Juxtanodin by phosphorylation and RhoA GTPase……………………………………………………………………… .111 4.5 Conclusions…………………………………………………………………… 115 4.6 Future studies……………………………………………………………………117 vii Table of contents REFERENCES……………………………………………………….119 viii References Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S.Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells. 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Curr Biol. 2003 Oct 14;13(20):1820-3. 144 [...]... hydrolyzed by the intrinsic ATPase activity of actin and the ADP -actin favors to dissociation from the pointed end of actin filaments, resulting in their disassembling The actin- depolymerizing factor (ADF) and the members of cofilin family play a critical role in actin filaments disassembling These ubiquitous and highly conserved proteins bind to ADP–F -actin and drive the dissociation of ADP -actin from filaments... directly interact with actin and this interaction was mediated by the C-terminal F -actin binding domain, which comprised the last 14 amino acid of JN Studies on the role of JN in actin dynamics showed that it could prevent F -actin depolymerization in vivo and in vitro, suggesting that JN was possibly involved in the F -actin based structures and behaviors of oligodendrocyte As expected, JN over-expression in. .. side of actin fibers 90 Figure 3.13 Juxtanodin inhibited cell migration through its C-terminal F -actin domain 92 Figure 3.14 Effect of T258 phosphorylaiton of Juxtanodin on cellular morphology and actin cytoskeleton of OLN-93 cells 94 Figure 3.15 Effect of S278 phosphorylaiton of Juxtanodin on morphology of OLN-93 cells 96 Figure 3.16 Effect of S278 phosphorylaiton of Juxtanodin on cellular actin cytoskeleton. .. functioning as a binding site for some actin- binding proteins (Dominguez, 2004) Actin can undergo conformational changes depending on the interaction with nucleotide, cations and actin- binding proteins and its polymerization state (Schüler et al., 2001) In the ATP-bound form, the conformation of actin confers its high affinity with other actin molecules which induces the self-association into two tightly intertwined... action of their severing actin filaments will be detailed discussed in the following section of Actin- binding proteins: actin filament severing proteins’ 12 Introduction 1.2.3.2 Actin filament extension, disassembly and stabilization Upon nucleated, actin filaments extend rapidly through addition of ATP–G -actin at the barded end The extension of filamentous actin is controlled partly by capping proteins... cytoskeleton of OLN-93 cells 98 Figure 3.17 Inhibition of RhoA magnified the arborization of OLN-93 cells induced by Juxtanodin x 100 List of abbreviations LIST OF ABBREVIATIONS aa amino acid Ab antibody ABC avidin-biotin conjugate ABPs actin- binding proteins ADF actin- depolymerizing factor AIP-1 actin- interacting protein 1 ALS amyotrophic lateral sclerosis AP alkaline phosphate Arp actin related protein BCA... capable of antagonizing gelsolin- and ADF/cofilin-mediated depolymerization by competitive binding to actin filaments (Kuhn et al., 2008) 1.2.4 Actin- binding proteins As mentioned in the above sections, actin dynamics in cells is precisely regulated by many actin- binding proteins, whose activities are under the spatiotemporal control of different signaling pathways Many actin- binding proteins are ubiquitously... pathfinding and epithelial folding, was also generated by the actin cytoskeleton (Pollard and Borisy, 2003; Pantaloni et al., 2001) Actin is present in two different forms: one is the monomeric globular actin (G -actin) and the other is the assembled filamentous actin (F -actin) Actin monomer consists of 375 amino acids in the proteins size of approximate 43 kDa (kilodalton) It comprises of two domains,... figures Figure 3.8 Juxtanodin increased F -actin content of OLN-93 cells 82 Figure 3.9 The effect of Juxtanodin in antagonizing F -actin disassembly of OLN93 cells induced by Latrunculin A 85 Figure 3.10 The effect of JN on arborization of OLN93 cells 86 Figure 3.11 The effect of JN on cell spreading of OLN93 cells 87 Figure 3.12 Juxtanodin induced the formation of actin fibers of OLN-93 cells and localized... different actin genes coding for at least 6 actin isoforms were found (McLean et al., 1990) Mammals have at least six actin coding genes, which are divided into three groups: α- β- and γ-actins The isoforms are present in cell-type and tissue specific manner Four isoforms of α -actin are predominantly expressed in muscle cells and the less acidic β- and γ -actin are mainly found in non-muscle cells (Vandekerckhove . ROLE AND REGULATION OF JUXTANODIN IN ACTIN CYTOSKELETON OF OLIGODENDROCYTE MENG JUN NATIONAL UNIVERSITY OF SINGAPORE 2010 ROLE AND REGULATION OF JUXTANODIN. actin- binding proteins ADF actin- depolymerizing factor AIP-1 actin- interacting protein 1 ALS amyotrophic lateral sclerosis AP alkaline phosphate Arp actin related protein BCA bicinchoninic. 3.1.6 Juxtanodin inhibited F -actin disassembly in vitro……………………………79 3.1.7 Juxtanodin increased F -actin content of OLN-93 cells………………………81 3.1.8 Juxtanodin inhibited F -actin disassembly induced