CHARACTERISATION OF THE RHO GTPASE TARGET DISHEVELLED ASSOCIATED ACTIVATOR OF MORPHOGENESIS (DAAM1) ANG SU FEN NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERISATION OF THE RHO GTPASE TARGET DISHEVELLED ASSOCIATED ACTIVATOR OF MORPHOGENESIS (DAAM1) ANG SU FEN (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements Acknowledgements First and foremost, I would like to thank my supervisor, Dr Ed Manser, for his mentorship and guidance in these four years. Also, I would like to thank Dr Jackson Zhao, my co-supervisor, who has offered me tremendous help, be it in experimental techniques or data analysis. I am also grateful to Prof Louis Lim for granting me a position in GSK-IMCB lab to conduct my research, which finally led to the generation of this thesis. At the same time, I would like to extend my gratitude to members on my thesis advisory committee, Dr Victor Yu and Dr Hooi Shing Chuan, for their suggestions and guidance that helped me progress through different stages of my project. I am most fortunate to be part of the GSK-IMCB lab, a fun and interactive lab that one would never want to leave. I would like to thank all my fellow lab mates for their support and presence in the lab which makes it a lively place to be in. Special thanks go to Elsa, Yeow Fong and Jet Phey, who provided me with much assistance when I first joined the lab and in a way, inducted me into the lab. Thanks also go to Yohendran, Delina, Praju, Jeff Yong, Paochun and Soon Tuck, fellow students who join me in the pursuit of a doctorate. I would also like to thank members of the lab (past and present) who have made my four years of stay most memorable: Sinnisky, Evonne, Charis, Christy, Puneet, Irene, Dr Jeff Robens, Dr Chan Wing, Dr Dong Jing Ming, Dr Perry Chan, Dr Thomas Leung, Dr Ivan Tan, Mr Joel Lee, Ms Rossiter and Ms Rani. Apart from my lab members, I also want to thank my best friends, Chengying, Jiamin, Aifen, June and Huifen, whom I have known for more than ten years. They -i- Acknowledgements never fail to offer encouragement and advice in times of need, which helped me overcome difficulties encountered in different stages of my life. I would also like to express my thanks to members in my Honours lab and my ex-classmates. They include Mirtha, Baohua, Koh Shiuan, Lily, Hang Yee, Kah Weng and Shiqi. I am also glad to have Boon Kiat, someone who have always been my source of comfort and support, for company in this arduous stage of thesis writing, In addition, I would like to express my thanks to my family, both immediate and extended, for being so understanding and encouraging in this whole process. Their never-ending support and unconditional care give me the strength to persevere till the end. Finally, I would like to thank A*STAR for giving me this scholarship, without which I would not be able to come this far, and also IMCB for providing the necessary facilities for my research. I would also like to thank NGS for providing their best support to students. It would be impossible to mention everyone here, so before I end I would like to say a big thank you to all those who have helped me in one way or another and have contributed to making this thesis a success. Thank you very much! - ii - Table of Contents Table of Contents Acknowledgements .i Table of Contents . iii Abstract .ix List of Figures and Tables .xi Abbreviations . xiii Chapter 1. 1.1. Introduction Formins 1.1.1. Formin family of proteins 1.1.2. Formin domains .2 1.1.3. Lessons from Diaphanous 1.2. Rho GTPases 10 1.2.1. Rho GTPase family 10 1.2.2. Regulation of Rho GTPases .11 1.2.3. RhoA, Rac1 and Cdc42 13 1.2.4. Rho GTPases in cell migration 16 1.3. Dishevelled Associated Activator of Morphogenesis (DAAM1) .17 - iii - Table of Contents 1.4. Thesis objectives 18 Chapter 2. Materials and Methods 20 2.1. Cell culture .20 2.2. Antibodies 20 2.3. Reagents .21 2.4. Plasmids and oligonucleotides .21 2.4.1. List of DAAM1 primers .22 2.5. Site-directed mutagenesis 24 2.6. Sequence analyses and alignment 26 2.7. Transfection .26 2.8. siRNA Transfection .26 2.9. Drug treatment .27 2.10. Lipid raft purification .27 2.11. Immunoprecipitation 27 2.12. Western analysis 28 2.13. Wound healing assays 28 2.14. Microinjection 29 - iv - Table of Contents 2.15. Immunofluorescence 29 2.16. Live-cell imaging .30 Chapter 3. 3.1. Results .31 DAAM1 .31 3.1.1. DAAM1 domains .31 3.1.2. DAAM1 isoforms 31 3.1.3. DAAM1 versus DAAM2 .34 3.1.4. DAAM1 in different species 34 3.1.5. Analysis of DAAM1 FH3 domain .34 3.2. Generation and purification of DAAM1 antibody .38 3.3. Localisation of DAAM1 38 3.3.1. Design of DAAM1 truncation constructs 40 3.3.2. Localisation of endogenous DAAM1 40 3.3.3. DAAM1 localisation to the actin cytoskeleton 40 3.3.4. DAAM1 and the Golgi apparatus 44 3.3.5. Localisation of putatively active DAAM1: DAAM1 FH1-FH2-DAD 44 -v- Table of Contents 3.3.6. The FH1-FH2-DAD from different formins produce similar phenotypes .49 3.3.7. The activity of DAAM1 in vivo requires a C-terminal basic region 54 3.3.8. Lipid raft association of DAAM1 59 3.4. Effects of DAAM1 on the cytokeleton 63 3.4.1. Effect of DAAM1 on actin 63 3.4.2. Effect of DAAM1 on microtubules .63 3.4.3. Effect of DAAM1 on myosin II .63 3.5. Interaction of DAAM1 with Rho GTPases 64 3.5.1. Co-immunopreciptation assays and Western analysis .64 3.5.2. Immunofluorescence 67 3.5.3. Deriving GTPase binding defective DAAM1 67 3.5.4. Localisation of DAAM1 N-ter, (1-233) and (1-440) GBD mutants 70 3.6. siRNA knockdown of DAAM1 in cell lines 73 3.7. DAAM1 and Dishevelled (Dvl2) 73 3.8. A system to test DAAM1 involvement in cell migration 75 3.8.1. DAAM1 is essential for Golgi apparatus orientation in cell migration .79 - vi - Table of Contents Chapter 4. 4.1. Discussion 90 DAAM1 localisation and its significance 90 4.1.1. DAAM1 localisation to actin stress fibres .91 4.1.2. DAAM1 localisation to the Golgi 92 4.1.3. DAAM1: To Golgi or actin stress fibres? 92 4.1.4. DAAM1 localisation with a subset of myosin II fibres .93 4.2. Defining regions important for DAAM1 activity in vivo 94 4.2.1. DAAM1 FH1-FH2-DAD and actin polymerisation 94 4.2.2. DAAM1 FH1-FH2-DAD and the HeLa phase-dark structures .95 4.2.3. DAAM1 FH1-FH2-DAD and microtubule reorganisation and stabilisation 96 4.2.4. DAAM1 FH1-FH2-DAD and lipid raft association 97 4.2.5. Contribution of different domains to activation .97 4.2.6. DAAM1 localisation: COS versus HeLa 99 4.3. Rho GTPases and DAAM1 99 4.3.1. Interaction of DAAM1 with GTPases .99 4.3.2. Analysis of DAAM1 GBD mutants .100 - vii - Table of Contents 4.4. DAAM1 and Dishevelled (Dvl2) 101 4.4.1. DAAM1 and Dvl2 localisation 102 4.4.2. DAAM1, Rho GTPase and Dvl2 association 103 4.5. Role of DAAM1 in polarised migration 103 4.6. A summary of DAAM1 localisation 106 4.7. Conclusion .108 Bibliography 110 - viii - Chapter 4: Discussion 4.7. Conclusion DAAM1 shares common features with formins in terms of domain structure. The regulation of DAAM1, however, may differ from that of typical formins since the Cterminal 33 residues (10/33 basic) is required for generation of long, fine actin fibres in HeLa cells, a phenomenon observed in other active formins. In mDia1, deletion of its C-ter region up to the DAD is reported to generate an active phenotype [37]. It would be interesting to compare directly the effects of DAAM1 with those of other formins. My results with DAAM1 is the first suggestion that the region C-terminal to the DAD positively contributes to formin function, and indeed in the case of DAAM1, is essential for in vivo effects. It would be of interest to test this with respect to Wnt signalling. DAAM1 can bind to active versions of RhoA, Rac1 and Cdc42. It seems likely that localisation of DAAM1 actually determines the type of Rho GTPase it interacts with and the connection to lipid rafts suggests segregation of DAAM1 signalling at the plasma membrane as for RhoA and Rac1 [61, 97]. The significance of the DAAM1 FH1-FH2-DAD enriched phase-dark patches in HeLa cells and colocalisation of Glu-tubulin and cholera toxin B needs to be tempered by the observation that caveolin-1 does not co-segregate with these structures (Figure 3.14d). HeLa cells appear to express some components that generate these structures but there is no clue as yet to its nature; it is only known that this process is not DAAM1specific. In COS cells, I have shown that DAAM1 knockdown resulted in an impairment of Golgi/MTOC orientation in wound assays. This places DAAM1 in the - 108 - Chapter 4: Discussion cell polarity pathway together with various players like Cdc42, Par6, and PKC . Since DAAM1 has been shown to mediate actin polymerisation and microtubule stabilisation, it is likely that DAAM1 is required for Golgi orientation because of its ability to drive cytoskeletal remodelling. Targeting of DAAM1 to the Golgi is GTPase independent but requires the first 44 residues whose function is not revealed by homology search. Wound assays performed in the presence of dominant inhibitory Cdc42N17 show strong inhibition of Golgi orientation as established [65]. Therefore, I favour the notion that DAAM1 interacts with GTP bound Cdc42 at the Golgi region in spite of the weaker biochemical interaction with this GTPase. Existing data suggests that DAAM1 is a complex multi-domain protein subjected to multiple levels of regulation. Future studies of DAAM1 should be greatly aided by the observations I have made here regarding the targeting and structurefunction relationships of the protein. If crystal structures of larger fragments of DAAM1 become available, some of the regulatory issues might be resolved. The role of N- and C-terminal residues to its localisation requires screening for potential interactors (both lipids and proteins). 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Effects of DAAM1 10 0-350 on myosin II in COS 7 cells 66 Figure 3 .17 Interaction of DAAM1 with Rho GTPases (Co-IP) 68 Figure 3 .18 Interaction of DAAM1 with active Rho GTPases (IF) 69 Figure 3 .19 Generation of DAAM1 N-ter GBD mutants and determination of binding to the Rho GTPases 71 Figure 3.20 Localisation of DAAM1 GBD mutants 72 Figure 3. 21 siRNA knockdown of DAAM1 74 Figure 3.22 DAAM1 and Dishevelled. .. site - xv - Chapter 1: Introduction Chapter 1 Introduction 1. 1 Formins The focus of this thesis is on Dishevelled Associated Activator of Morphogenesis 1 (DAAM1) , a member of the formin family of proteins The name formin originated from the discovery of the limb deformity gene in mice This gene was found to be important for the formation of the limbs and kidneys, hence was given the name formin Mice... the biochemistry of DAAM1, the protein that is the focus of this thesis Habas et al identified DAAM1 as an interactor of the PDZ domain of mouse Dishevelled 2 (Dvl2) by yeast two hybrid [ 81] DAAM1 is placed downstream of Wnt signalling as DAAM1 is required for interaction of Dvl with RhoA upon Wnt stimulation The Dvl-RhoA-DAAM1 complex is proposed to be essential for activation of RhoA Use of DAAM1... endogenous Rho activation 1. 2.3 RhoA, Rac1 and Cdc42 RhoA, Rac1 and Cdc42 are the most well studied members of the Rho GTPase family They have been shown to be involved in various signalling pathways, many of which involve the cytoskeleton There are three members in the RhoA subfamily, RhoA, RhoB and RhoC RhoA has been shown to bind to downstream targets like Rhokinase (ROK/ROCK) [55, 56] and mDia1 [36]... In the case of the fission yeast, sequences within the FH3 of Fus1p targets it to the projection tip during conjugation [17 ] The C-terminal portion of the FH3 in mDia1 is required for its localisation to the mitotic spindle in HeLa cells [18 ] Given the absence of well defined residues and the lack of functional data, the existence of a FH3 “domain” is speculative -5- Chapter 1: Introduction The FH1... [11 ], many studies have been performed to investigate its structure and function Biochemical data from Watanabe et al supports the binding of endogenous mDia1 to activated RhoA and profilin [11 ] The interaction of mDia1 with profilin was independent of RhoA binding and was shown in later studies to be mediated by the FH1 domain [36] The FH1 and FH2 domains of mDia1 are both required for formation of. .. pathways generates the spatial and temporal delivery of components for movement of the cell [79, 80] Cdc42: directional sensing & polarity Rho: contractility Golgi Nucleus Rac: lamellipodia Rho: protrusions Trailing edge Leading edge Figure 1. 6 Role of Rho GTPases in cell migration - 16 - Chapter 1: Introduction 1. 3 Dishevelled Associated Activator of Morphogenesis 1 (DAAM1) Most studies of formins have... the lipid tails of the GTPases An activating signal is required to trigger the release of Rho GTPases from the GDIs and allow their translocation to the membrane for another round of activation Cycling between the “ON” and “OFF” states ensures that the Rho GTPases function as efficient switches in cellular signalling where fast activation and termination of the signal is required (Figure 1. 5) GTP Rho- GDP... [5] The structure of the GBD with either the DAD or RhoC was also resolved for mouse Diaphanous (mDia) [6 -10 ] These successes in protein structure resolution helped substantiate the -1- Chapter 1: Introduction proposed structural organisation of various domains and the mechanism of formin regulation and function Dia2 FMN1 DAAM1 DAAM2 Dia3 Dia1 FMN2 FRL2 Delphilin FRL3 FRL1 FHOD1 INF2 FHOD2 INF1 Figure . Rho GTPases 10 1. 2 .1. Rho GTPase family 10 1. 2.2. Regulation of Rho GTPases 11 1. 2.3. RhoA, Rac1 and Cdc42 13 1. 2.4. Rho GTPases in cell migration 16 1. 3. Dishevelled Associated Activator of. Chapter 3. Results 31 3 .1. DAAM1 31 3 .1. 1. DAAM1 domains 31 3 .1. 2. DAAM1 isoforms 31 3 .1. 3. DAAM1 versus DAAM2 34 3 .1. 4. DAAM1 in different species 34 3 .1. 5. Analysis of DAAM1 FH3 domain 34. Chapter 1: Introduction - 1 - Chapter 1. Introduction 1. 1. Formins The focus of this thesis is on Dishevelled Associated Activator of Morphogenesis 1 (DAAM1) , a member of the formin family of