Investigating the role of a dynamin-actin interaction By: Sarah Elizabeth Palmer A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Science Department of Biomedical Science November-2015 Abstract Endocytosis is a fundamental cellular process which facilitates the uptake of lipids and proteins from the plasma membrane. During endocytosis, the plasma membrane is internalised through the formation of an invagination, which is then pinched off to form a vesicle. Endocytosed vesicles are then able to fuse with endosomal compartments allowing cargo to be sorted within the endo-lysosomal system. One of the most well characterised forms of endocytosis is clathrin mediated, which takes place in distinct stages. These include coat and clathrin recruitment, membrane invagination and finally scission which releases the vesicle into the cytoplasm. Cells require many different molecules to orchestrate each stage including the GTPase dynamin and cytoskeletal protein actin. Dynamin facilitates the scission stage of endocytosis where it oligomerises around the vesicle neck and, through the hydrolysis of GTP, enables scission. Furthermore dynamin-1 has been reported to bind directly to actin. In Saccharomyces cerevisiae (yeast) actin is essential to endocytic invagination in order to overcome turgor pressure. Yeast contain a dynamin-like protein Vps1 which acts during endocytic scission and has orthology with dynamin-1. In this thesis the direct interaction between Vps1 and actin was explored to elucidate how this interaction may be required during endocytosis. It is understood that both dynamin and actin are involved in other cellular processes and therefore the knowledge gained from investigating this interaction during endocytosis may well provide new insight into how these two molecules work together in other molecular systems. Thus, mutations found to perturb the Vps1-actin interaction were created in human dynamin-1 and preliminary results suggest this could have an effect on mammalian cell endocytosis and cell migration. This project has identified a novel Vps1-actin interaction which is required for the scission stage of yeast endocytosis. It also describes a point mutation in Vps1 E461K, which has been found to cause an early stage endocytic defect. I Acknowledgements I would like to take this opportunity to thank everyone who has helped me complete this thesis whether it be through your teaching, advice or support. Every single one of you have aided my progression through the great times, and the not so great times, to make this a very memorable and enjoyable few years. Firstly I would like to extend my immense thanks to Kathryn Ayscough who has supervised this project, taught and guided my ideas. She has always had time for me, even during the busiest of weeks and I am very grateful for her support. I would like to thank Steve Winder who has also supervised this project, taught me cell culture techniques and put up with me randomly turning up in his office to ask him questions out of the blue. My advisors Andy Furley and Liz Smythe have also been invaluable to my experience at this university. Not only for helping with my PhD progression but also with advice for my future career. They have taken the time to encourage and support my future plans and have been instrumental to my development throughout this degree. I would like to thank everyone in the Ayscough/Winder labs for making my time at Sheffield enjoyable both in and out of the lab. Specifically, I would like to thank Ellen Allwood and Iwona Smaczynska-de Rooij for teaching me so many different techniques, to Ellen for being there to troubleshoot experiments (amongst other things!) and to Iwona for all your help with the publications. I would also like to especially thank Chris Marklew and Laura Jacobs for keeping me going and discussing ideas, Joe Tyler, Kate Collins-Taylor and Daniel Leocadio Victoria for many chats and lots of fun. To Aga Urbanek for your excellent attitude and never putting up with any rubbish! And to Lemya Abugharsa, Laila Moushtaq, Emma Hoffman, Tracy Emmerson and Katja Vogt-who is continuing this project- I will miss you all. Thanks is also due to Rob Piggott who guided me through my nervous first year and introduced me to PhD life. Since leaving the lab Rob has taken the time to proof read my thesis and provide excellent feedback and for that I am very grateful. Thanks to the ‘Unkillable Clerics’ Wezz Booth, Dave Pickthall, Ryan O’Conner, Diz Carberry and Ash Tuck for introducing me to post-rock music and getting me back in to ‘cello playing. Thanks also to Dom Kolarova and Craig Fishwick for all those hikes in the peaks and trips to the pub which provided me with many well needed breaks from work. Finally I would like to thank my family, Mum, Dad, Helen, Steph and my Grandad Arthur for putting up with me disappearing for four years to complete this project. You have always believed I could do it even when I didn’t, thanks for always being there. Similarly I would like to thank Sid Dongre. You have given me the confidence to complete this thesis and your continual support means the world to me. II Contents Abstract …………………………………………………………………………………………………………………………..…..I Acknowledgments ……………………………………………………………………………………………………………...II Contents…………………….…………………………………………………………………………………………………..……1 List of figures ...................................................................................................................... 7 List of Abbreviations ......................................................................................................... 11 Chapter 1. Introduction ..................................................................................................... 13 1.1 Endocytosis ....................................................................................................................... 14 1.1.1 Clathrin mediated endocytosis ................................................................................... 16 1.1.2 Yeast clathrin mediated endocytosis ......................................................................... 17 1.1.2.1 Stage 1: Coat assembly and nucleation of a clathrin coated pit ............................. 20 1.1.2.2 Stage 2: Invagination of a clathrin coated pit ......................................................... 21 1.1.2.3 Stage 3: Scission and vesicle release ....................................................................... 22 1.1.3 Mammalian CME ....................................................................................................... 23 1.2 Actin .................................................................................................................................. 26 1.2.1 G-actin structure ........................................................................................................ 28 1.2.2 F-actin structure ......................................................................................................... 28 1.2.3 Actin polymerisation .................................................................................................. 29 1.2.4 Actin structures in yeast ............................................................................................. 30 1.3 Actin in endocytosis .......................................................................................................... 32 1.3.1 Actin requirement in yeast CME ................................................................................. 32 1.3.2 Actin in mammalian CME ........................................................................................... 33 1.4 Dynamin ............................................................................................................................ 35 1.4.1 Domains of dynamin .................................................................................................. 35 1.4.2 Dynamin function at the scission stage of endocytosis ............................................. 38 1.4.3 Dynamin isoforms 1, 2 and 3 ..................................................................................... 39 1.4.3.1 Dynamin-1 ............................................................................................................... 39 1.4.3.2 Dynamin-2 ............................................................................................................... 40 1.4.3.3 Dynamin-3 ............................................................................................................... 41 1.5 Dynamin and actin in cellular processes ........................................................................... 42 1.5.1 The direct dynamin-actin interaction ......................................................................... 42 1.5.2 Dynamin and actin in endocytosis ............................................................................. 43 1.5.2.1 The function of dynamin and actin in CME ............................................................. 43 1.5.2.2 Dynamin and actin in clathrin independent endocytosis ........................................ 44 1 1.5.3 Interactions of dynamin with actin binding proteins .................................................. 45 1.5.4 The role of dynamin and actin in cell migration ......................................................... 46 1.5.5 Dynamin and actin in podosomes and invadopodia ................................................... 47 1.5.6 Dynamin in actin comet tails .....................................................................................