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Download Thesis This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Characterisation of the Formin Protein FHOD1 in Striated Muscle Dwyer, Joseph Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Characterisation of the Formin Protein FHOD1 in Striated Muscle Joseph Dwyer Thesis submitted to King’s College London for the degree of Doctor of Philosophy June 2015 1 “I never practice; I always play.” ~ Wanda Landowska "The saddest aspect of life right now is that science gathers knowledge faster than society gathers wisdom." ~ Isaac Asimov “One becomes firmly established in practice only after attending to it for a long time, without interruption and with an attitude of devotion.” ~ Yoga Sutra I.14 This thesis is dedicated to my parents: Dr. John P. Dwyer and Mrs. Angela Micioni Dwyer Their love and support have helped guide me through my studies and I will always be grateful to them. 2 Acknowledgements I would like to thank the following people for their help and support. My first supervisor Dr. Elisabeth Ehler for her help and support throughout my research studies. Her enthusiasm and kindness have provided me much of the motivation and inspiration for my work. My second supervisor Prof. Anne Ridley for her helpful discussions and for the donation of expression constructs. Dr. Thomas Iskratsch for the donation of expression constructs and general support. Without his help in learning the relevant molecular biology techniques this project would not have been possible. Dr. Sue Perera for her invaluable help in learning GST pull-down assays, for general lab support, and for her friendship. Dr. Atsushi Fukuzawa for his support in the lab with the yeast two- hybrid technique. Dr. Ay Lin Kho for her assistance in the neonatal rat cardiomyocyte preps. I would like to extend a very warm thanks to Ms. Birgit Brandmeier for her general lab support and for her friendship. The current and previous members of Dr. Elisabeth Ehler’s group for help and support in the lab: Dr. Thomas Randall, Dr. Sagair Hussain, Ms. Marlene Plüß, Ms. Nadine Gose, and Ms. Nadine Lohmann. The current and previous members of Prof. Mathias Gautel’s group for their help and support in the lab: Prof. Mathias Gautel, Dr. Alexander Alexandrovich, Dr. Martin Rees, Mr. Christopher Jenkins, Dr. Mark Holt, Mr. Andrea Ghisleni, Dr. Sarah Waters, and Dr. Elena Rostkova. Dr. Daniel Soong and Dr. Peter Stevenson for their assistance with the confocal microscopes. I would also like to thank Dr. Pauline Bennett and Ms. Amanda Wilson for their helpful discussions regarding the intercalated disk. From the group of Dr. Maddy Parsons I would like to thank Dr. Asier Jayo, for advice on performing F- actin quantification in cells and Ms. Giulia Villari, for many in depth scientific discussions and for her kindness and friendship. The Randall Protein Production Facility for their assistance with cloning: Dr. Paul Brown, Mrs. Renée Tata, Dr. Mitla Garcia-Maya. I would also like to acknowledge the memory of Henrietta Lacks and her contribution to science in the creation of HeLa cells. I would like to thank all of my friends in the Randall Division for their help and support throughout my studies, especially, Ms. Ivanka Sevrieva, Ms. Maira Silva, Dr. Thomas Kampourakis, Dr. Andrea Knowles, Ms. Rumena Begum, Ms. Upamali Perera, Ms. Rachael Inglis, Mr. 3 Richard Downes, and Ms. Louise Moyle. I would like to give a special thanks to Dr. Sehrish Rafique, Ms. Kaye Batten, and Mrs. Stefanie Hoffart for their love and support. I would also like to thank Ms. Francesca Ludwinski for her friendship and support during all of my scientific studies. I would like to give a very warm thanks to Ms. Marie Lecomte who has provided me with a decade of friendship and the inspiration to study science. Lastly I would like to give the biggest thanks to my family: Dr. John P. Dwyer, Mrs. Angela Micioni Dwyer, Ms. Francesca Dwyer, and Jack Dwyer. I would also like to thank my grandmother Mrs. Thea Stanchieri Micioni and Dusty Dwyer who are sadly no longer with us but will remain in my heart forever. My family are my biggest inspiration and provide me the strength and will to excel in every aspect of life. Sources of Funding: Thank you to King’s College London and the British Heart Foundation for their funding, help, and support. Thank you to the Randall Division of Cell & Molecular Biophysics and the BHF Centre of Research Excellence for their help and support. 4 Abstract I Abstract Formin homology 2 domain containing protein 1 (FHOD1) is a diaphanous related formin of the FHOD subclass. In a similar manner to other formins, FHOD1 has primarily been found to regulate the polymerisation of actin-based structures in cells. In muscle, actin is a major constituent of both skeletal and cardiac myocytes, with the thin filament system of sarcomeres and the cytoskeleton being partly composed of actin. FHOD proteins have previously been highlighted as important regulators of muscle cell biology since FHOD3, a close relative of FHOD1 was shown to be essential for myofibrillar maintenance. There is little known about the regulation of actin-based structures in muscle cells. We therefore aimed to characterise FHOD1 and probe into its involvement in myofibrillar and cytoskeletal regulation. In this study of FHOD1 we addressed various aspects of the protein including its expression pattern and localisation, function, regulation, and novel interacting partners. Insight into the expression pattern of FHOD1 was gained by examining relative protein levels in different tissues, including both healthy and diseased heart. Subcellular localisation was addressed in a number of fluorescence microscopy experiments through antibody localisation studies in muscle tissue and cultured cells and through transient transfection of GFP tagged constructs. Expression of GFP tagged fragments and mutants helped to delineate the functional distribution of the FHOD1 molecule in cells. The function of the protein was further probed via molecular knockdown by RNAi and by looking at the capacity of FHOD1 to polymerise actin in cells. The role of formins in the heart was more broadly addressed by drug inhibition of their actin polymerising capacity. Previous studies have suggested that the Rho family of small GTPases as well as the Src kinases regulate FHOD1. Involvement of the GTPases and Src was shown through biochemical experiments. Finally, a number of Yeast2Hybrid assays were performed using different domains of FHOD1 to screen for novel binding partners. Our findings would suggest that FHOD1 is a crucial regulator of the myofibrillar apparatus and the cytoskeleton at the level of actin in striated muscle. 5 Table of Contents II Table of Contents I Abstract ........................................................................................................................... 5 II Table of Contents .......................................................................................................... 6 III List of Figures ............................................................................................................ 13 IV List of Tables ............................................................................................................. 18 V. Abbreviations ............................................................................................................. 19 1. Introduction ................................................................................................................. 26 1.1 Muscle ................................................................................................................... 26 1.1.1 Smooth Muscle ............................................................................................... 28 1.1.2 Cardiac Muscle ............................................................................................... 29 1.1.3 Skeletal Muscle ............................................................................................... 31 1.1.4 Contractile Mechanism of Striated Muscle .................................................... 32 1.2 The Sarcomere ....................................................................................................... 33 1.2.1 The Z-disk ......................................................................................................
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