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An Investigation Into the Physiological Role of Annexin A11

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An Investigation into the Physiological Role of Annexin A11 Luxmi Fatimathas A thesis submitted to University College London for the degree of Doctor of Philosophy September 2009 Division of Cell Biology UCL Institute of Ophthalmology 11-43 Bath Street London EC1V 9EL -1- Declaration I, Luxmi Fatimathas, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Luxmi Fatimathas -2- Abstract The calcium-dependent phospholipid-binding protein annexin A11 was discovered over 25 years ago, however little is known about its function. This thesis supports a role for annexin A11 in the cell cycle and suggests an association with the microtubule network. Recent studies have implicated annexin A11 in the autoimmune disease sarcoidosis, which is further investigated in this study. A single nucleotide polymorphism within annexin A11 (R230C) was identified as a highly associated susceptibility locus for sarcoidosis. Over-expression of annexin A11WT and annexin A11R230C showed no difference in their distribution. Stimulation with ionomycin, which induces a rise in intracellular calcium, resulted in the translocation of both variants to the plasma membrane and nuclear envelope with approximately the same time course. The calcium-dependent translocation of annexin A11 is therefore unaffected by the R230C mutation. In this one aspect there appears to be no difference between the two variants, however much still remains to be investigated such as the potential extracellular roles of these variants and their function in immune cells. Much of the work in this thesis has focused on elucidating the function of wild type annexin A11, particularly during the cell cycle. During mitosis the distribution of annexin A11 is highly dynamic and localises to the mitotic spindle at metaphase and anaphase, as well as the midbody at cytokinesis. The use of methanol fixation highlighted the co-localisation of annexin A11 with the microtubule network throughout mitosis, as well as at interphase. Furthermore annexin A11 was shown to concentrate at the centrosome, again during both mitosis and interphase. Several centrosomal proteins also localise to the midbody and are key regulators of mitotic progression, supporting a similar role for annexin A11. Furthermore the centrosomal localisation of annexin A11 may serve as an ideal docking site from which it can be easily targeted throughout the cell. This study highlights the novel localisation of annexin A11 to the centrosome and the microtubule network. Furthermore these findings contribute to a growing picture for the role of annexin A11 in the cell cycle, particularly with regards to its use of the microtubule network – a feature which may also have a role in interphase cells. -3- For Amma and Appa Thank you for everything -4- Acknowledgements I would like to start by thanking my supervisor Prof. Stephen Moss for giving me the opportunity to pursue a PhD in his lab and for the guidance he has given me over the past three years. This PhD would not have been possible without the support of my PhD programme at the LMCB and funding provided by the MRC. I would also like to thank my thesis committee; Prof Karl Matter, Dr Paul Kellam and Dr Adolfo Saiardi, for their continued encouragement and reassurance. The daily work of this PhD has been made infinitely easier through the efforts of Steven Griffiths, Claire Cox and Aida, who have kept the lab running smoothly over the past few years. There are so many people at the Institute who have all made my time here such great fun. So a lot of thank yous go to; Ian „Dr Evil‟ White, Emily Eden for being my favourite B.F.G, Veronika, James „n‟ Katy, my steadfast crossword buddy Rush, Marcin, Hideki and Emily Steed for being the smiliest person I know. A huge thanks goes to Becca, my super post doc. Thank you for teaching me everything I know about molecular biology, for never making me feel stupid even when I didn‟t have a clue (!) and for always being so supportive, both in and out of the lab. Matt, my darling work hubby, thank you for your inexhaustible enthusiasm for science and for being my verbal sparring partner! I wouldn‟t have been able to get through this PhD were it not for my lab and office. Jenny and Jay, I‟m so glad you both chose to join the Institute, you‟ve not only made my time at the Institute more fun but have made all our LMCB trips something to be cherished. Thanks to Mark „Bourbon‟ Evans for being the ever reliable voice of reason and Adam for being a God. Ah-Lai, you‟ve seen me through so many ups and downs, you‟ve always seen the best in me and been there to lend a patient ear – thank you so much. Thanks also to all you Mullers out there, both past and present. Hari, Christina, Aaron and Megan – you‟ve all made lunch a real laugh and provided much appreciated banter. A special thanks to Bhatia; from clueless undergrads to hopefully not so clueless postgrads, you‟ve always been my constant friend. Thanks for never being too busy to be bugged and enduring my often nonsensical air filling! To Shweta, my unwarranted target for all things insulting and sarcastic - that you faithfully endure that deserves thanks enough! Thank you for your many shoulder-leaning, rock-being services and thanks to both you and Anand for making Cambridge an ever relaxing sanctuary amidst a crazy three years – I am most grateful. I would also like to thank Shazeen for being truly understanding and for our many corridor chats! -5- And to Rugina for always being honest but supportive and making sure I stayed on track. I am also endlessly appreciative to Michelle, Nicola and Saj for keeping me relatively sane and reminding me of life outside of the lab. Lastly I want to thank my Appa for setting the foundations of my education from day one, without which I never would have made it this far. Thanks also to my Amma for your unwavering support and for giving me the strength and courage to follow this road. -6- Contents List of Figures .................................................................................................................. 10 List of Tables ................................................................................................................... 13 List of Abbreviations ........................................................................................................ 14 Chapter One: .................................................................................................................. 17 1. Introduction .................................................................................................................. 18 1.1 The Annexin Family ................................................................................................................. 18 1.1.1 Annexin Nomenclature and Molecular Phylogeny ............................................................... 18 1.1.2 Annexin Structure ................................................................................................................. 20 Annexin Core Domain ................................................................................................................ 20 Annexin N Terminal Head .......................................................................................................... 22 1.1.3 Annexin Interacting Proteins ................................................................................................ 24 S100 Proteins ............................................................................................................................ 24 Cytoskeletal Proteins ................................................................................................................. 25 Other Ligands ............................................................................................................................ 26 1.1.4 Annexins in Membrane Trafficking ....................................................................................... 27 Annexins in Exocytosis .............................................................................................................. 28 Annexins in Endocytosis ............................................................................................................ 29 1.1.4 Annexins in Membrane Organisation ................................................................................... 32 1.1.5 Annexins and Ion Channels ................................................................................................. 33 1.1.6 Extracellular Roles of the Annexins ...................................................................................... 35 1.1.7 Annexins in Disease ............................................................................................................. 37 Cancer ....................................................................................................................................... 37 Diabetes ..................................................................................................................................... 38 Autoimmune Diseases ............................................................................................................... 39 1.2 Annexin A11 ............................................................................................................................ 39 1.2.1 Primary Structure .................................................................................................................
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