Tropomyosins and the actin cytoskeleton in neuronal morphogenesis and differentiation A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy (Medicine) at the University of New South Wales, 2011 Nikki Curthoys Neurodegeneration and Repair Laboratory Oncology Research Unit School of Medical Sciences Faculty of Medicine University of New South Wales This thesis is dedicated to my mum Jan Curthoys 09.07.1944 – 28.06.2003 Strength and grace And my nan Joan Margery Curthoys 23.03.1923 – 12.08.2010 A warm sunbeam iii Thesis Abstract The actin cytoskeleton is crucial for many functions including cell motility, cytokinesis, and vesicle formation. Tropomyosins (Tm) are actin associated proteins which regulate the functional capacity of actin filaments. At least 40 Tm isoforms are produced from four genes (αTm, βTm, γTm, and δTm), with differential expression and localisation in tissues, cells and subcellular compartments. As some Tms are potential targets for anti- cancer therapies, one aim of this project was to measure how eliminating one subset of isoforms affected expression of other Tms in brain. Using a knockout (KO) mouse model lacking the γTm-gene 9d exon, regional distributions of Tms were investigated by Western blotting in wild type and 9d KO adult mouse Whole Brain, Cerebellum, Amygdala, Hypothalamus, Cerebral Cortex, Hippocampus, and Olfactory Bulb. KO of γTm-gene isoforms Tm5NM1 and Tm5NM2was shown to induce upregulation of other γTm-gene products. Regional expression patterns of other Tms, including the δTm-gene product Tm4, were established in brain. A previously unidentified product immunoreactive with the Tm4 antibody was observed and characterised as having similar biochemical properties to Tm4; 2D gel electrophoresis indicated Tm4 and this associated product were both post translationally modified. To investigate Tm4 function in neural cells, rat neuroblastoma cells (B35) were stably transfected with a mammalian vector containing the Tm4 gene. These cells were compared with previously developed B35 clones overexpressing the neuronal isoforms: TmBr1, TmBr2, or TmBr3. Using light microscopy it was shown that while overexpression of these isoforms each induced neurite outgrowth, each had highly specific effects on neurite morphology. Analyses of TmBr2 and Tm4 overexpressing B35 cells by fluorescence activated cell sorting indicated these isoforms could significantly affect cell cycle exit. Quantitative proteomics approaches (LC-MS/MS and iTRAQ) identified proteins affected by iv overexpression of Tm5NM1, TmBr2, TmBr3, or Tm4 isoforms in B35 cells. These data show for the first time that Tms differentially induce changes in the levels of many other proteins, and Tm expression results in isoform specific profiles of other actin binding proteins (ABP). The main aims of this work were: 1) to investigate the potential of Tms to compensate for Tm isoform loss in brain, 2) to characterise a previously unidentified Tm associated product, and 3) to examine the role of Tms in neural morphogenesis, and the capacities of different Tm isoforms to regulate protein levels of other ABPs. The actin cytoskeleton underpins neurite outgrowth and branching, and the here examined Tms have been identified as regulators of these events and ABP expression. v Acknowledgements The Germans have a word meaning “friends who have been chosen as family”. Shame I don’t know what that word is. Thomas Fath has been a supervisor I was lucky to have; one who taught me really a lot about biochemistry and cell biology. And Germans. And also the more subtle (nowadays sadly overlooked) finer points of high German culture. Like their interesting attitudes to social graces, and what constitutes them. Thomas has also become family to me. Thomas, you and I know what a journey this thing was. I can’t imagine having done it with anyone else. Peter Gunning: Champion. What is it like to deal with students, year in, year out, and impress on them a certain way of approaching problems, a way of thinking, and a way of communicating data? I always found our talks helpful, insightful, and a pleasure. Meetings in your office felt like several species of small furry animals gathered together in a cave and grooving.. Justine Stehn: Thank you for being my unofficial supervisor. I have considered you a mentor throughout my time in this project, and I will continue to value your opinion highly. Andrea Connor- just as Wallerawang provides power for Sydney, you came in and put so much energy into this ordeal (.. you like that?). Some days even in farm jeans and goatkicker boots. Hope to be seeing you soon, mate. Hannah Freittag, you gave your all to the project; I almost forgive you for speaking German (i.e. throat-clearing) with Thomas so much in the lab. I know I will be reading your papers soon. Mel Desouza. Thanks for your help with this work; good to hear that you’re starting a PhD in the lab. Galina Schevzov, I am indebted to you not only for teaching me some of the lab fundamentals, but also laying so much groundwork for tropomyosin research. Much gratitude to all the lab folk who helped throughout this project: Claire Martin, Anne Poljak, Kim Guven, Marcus Beuke, Geraldine O’Neill, Christine Lucas, Jessie Zhong, Teresa Bonello, Anders Darhed, Jocelyn Widagdo, Adam Winterhalter and others..... And all the moral supporters- Cuc, Munther, James, Trevor, Seb, Em, Leonie, Mat,Franz, Fei, Abbey, Helen, Maha, Keerthi, Jayne, Helen, Marko, and Lawrence. Especially big thanks and much respect to my rockclimbing friends, Dave, Su Li, Steph, Martin, Digi, Stu, Enmoore, Chuin Nee, John, Albear, Kim and Glen. You have each taught me many valuable lessons about focus and persistence. vi Julie Ward and Renee Szokolai - legends. Even on those lowest of days just seeing you do my paperwork for me was enough to remind me to be thankful I’m not in admin. Thanks also for providing nutrition when things were dire. I know I owe you both. Jeff Hook, you are a gene guru, and a dead set lovely guy. I won’t forget how much time you’ve given me through this. All my best wishes for a bright future. Steve Palmer and Romain Barres, cheers to you both for showing incredible patience when explaining molecular biology to me. Edna Hardeman– my gratitude for all your support of this project and myself, and for your sage advice. Anthony Kee, you are a proofreading, paper recommending machine. I appreciate all your comments on the manuscript greatly. It’s been nice to have someone else in the lab who gets excited about the same things I do. Antonio Lee, hope to see you in Korea one day. We can unearth some Kim Chi together (I'll bring the pickles). Thanks for being a dude through the oddness. Sam Hess. Mate. Gratitude cubed for all of your time and help. You have given me something to be justly enthusiastic about. Delighted you are now the father of a bouncing Australian capital city, as well as FPALM. Dad, you have shown me such complete support, and have helped me so much through this and everything. You and your work set a standard to which I aspire. I am proud to be your daughter. Sasha, my sister: you proofread this whole entire thesis brick, and as such you deserve a round of applause. And heaps more. Sis, consider this acknowledgement a coupon for a one time chit you can call in for anything, anytime. Finally, my husband, Twig. You have tolerated all that weird postgrad stuff: the drifting off, the quiet cursing, the stupid hours science-making.. and through it all you have loved me. I truly am lucky to be your wife. vii Publications arising from this thesis Curthoys, N.M., Gunning, P.W., and Fath, T. (2011). Tropomyosins in Neuronal Morphogenesis and Development. In: Cytoskeleton of the Nervous System, R.A. Nixon, and A. Yuan, eds. (Springer New York), pp. 411-445. Fath, T., Chan, Y.-K. A., Vrhovski, B., Clarke, H., Curthoys, N.M., Hook, J., Lemckert, F., Schevzov, G., Tam, P., Watson, C.M., Khoo, P.-L., and Gunning, P. (2010). New Aspects of Tropomyosin-Regulated Neuritogenesis Revealed by the Deletion of Tm5NM1 and 2. European Journal of Cell Biology 89(7): 489-498. viii Selected abstracts Curthoys, N. M., Freittag, H., Schevzov, G., Gunning, P., and Fath, T. Tropomyosins and the Actin Cytoskeleton in Neuronal Morphogenesis and Differentiation. Invited Speaker, Queensland Brain Institute Special Seminar, Brisbane, Australia. 2010, December. Curthoys, N. M.,Schevzov G., Gunning P., and Fath T. The Tropomyosin Isoform Tm4 Affects Neuronal Morphogenesis and Dif ferentiation. Symposium speaker, OzBio International Conference, Melbourne, Australia. 2010, September. Curthoys N. M., Schevzov G., Gunning P., and Fath T. The Tropomyosin Isoform Tm4 Promotes Neuritogenesis and Neuronal Morphogenesis. Australia New Zealand Society for Cell Biology NSW Chapter Conference, Sydney, Australia. 2010, March. Curthoys N. M.,Schevzov G., Gunning P., and Fath T. Tropomyosin 4 Affects Neuronal Morphogenesis and Differentiation. American Society for Cell Biology Conference, San Diego, USA. 2009, December. Curthoys, N. M., Connor, A., Schevz ov, G., Gunning , P., F ath, T. Tropomyosin 4 Affects Neuronal Morphogenesis and Differ entiation. Speaker, Univ ersity of S ydney Discipline of Paediatrics and Child H ealth Conference, Sydney, Australia. 2009, August. Curthoys, N. M., Connor, A., Schevzov, G., Gunning, P., Fath, T. Defining the Repertoire of Tropomyosins in Adult Mouse Brain. Australian Society for Medical Research Conference, Sydney, Australia. 2009, June. Curthoys, N. M., Connor, A., Schevzov, G., Gunning, P., Fath, T. Defining the Repertoire of Tropomyosins in Adult Mouse Brain. University of Sydney School of Public Health Conference, Blue Mountains, Australia. 2008, November. Curthoys, N. M., Connor, A., Schevzov, G., Gunning, P., Fath, T. Defining the Repertoire of Tropomyosins in Adult Mouse Brain.