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Characterisation of Two Class V Myosins in the Fission Yeast Schizosaccharomyces Pombe

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Characterisation of Two Class V Myosins in the Fission Yeast Schizosaccharomyces Pombe Characterisation of Two Class V Myosins in the Fission Yeast Schizosaccharomyces pombe Them Zaw Win A Thesis Submitted to the University of London for the Degree of Doctor of Philosophy October 2000 Department of Biology University College London Gower Street London WCIE 6BT ProQuest Number: U643836 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643836 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 for mum and dad Acknowledgements First and foremost I would like to thank the most important person in my research study my supervisor. Prof. Jerry Hyams. I am indebted to Jerry for his guidance, support, fairness and encouragement throughout this project. The one thing I would like to thank him most for, is his faith in me for which my heart felt and deepest gratitude goes out to Jerry and for which I will always remember. I would also like to thank Dr Yannick Gachet for encouraging me to carry out important experiments and in particular teaching me how to do them. My thanks to Dr Daniel Mulvihill for teaching me how to perform certain experiments efficiently so that results can be obtained quickly. I would also like to express my gratitude to Dr Karen May for showing me how to carry out a Southern blot analysis and giving me support and motivation during my first year of research in Jerry’s lab. My thanks to Mrs Vasanti Amin for teaching me immunofluorescence techniques and for her support throughout my research study. My thanks also goes to two former project students under my supervision, Panagiota Loh and Nalini lyanger, for helping me in the localisation of Myo52 in fission yeast. Finally, I would like to express my warm and humble gratitude to mum, dad, my brother and sisters and to all my teachers for filling my life with love, kindness and guidance. ABSTRACT The fission yeast Schizosaccharomyces pombe is a cylindrically-shaped cell which grows by tip elongation and divides by medial fission. The actin cytoskeleton is intimately associated with both of these processes. The actin-activated motor protein, myosin, is involved in cellular processes that require the fimction of the actin cytoskeleton. S. pombe has five myosin genes spread amongst three different myosin classes. This thesis describes the identification and characterisation of two of these genes, myo51^ and myo52^^ both of which belong to the class V group of the myosin superfamily. Gene inactivation studies reveal that myoSlA cells display a phenotype that is indistinguishable from wild type whereas myo52A cells are temperature-sensitive with a rounded and pear-shaped morphology. The myo51A myo52A double mutant has the same characteristics as themyo52A single mutant. Overexpression of myo5I^ produce elongated cells with multiple nuclei whereas overexpression of myo52^ produce branched cells with thickened septa. Preliminary localisation of Myo51 shows that the protein is localised to one of the cell poles. Detailed localisation studies of Myo52 shows that the protein is localised to the actin-rich poles during tip growth and is present at the equator in cells undergoing cytokinesis. The localisation of Myo52 is dependent on an intact actin cytoskeleton. Further analysis of myo52A shows that the null mutant is resistant to cell wall digestion by Zymolyase. Immunoblot analysis of the a-glucan synthase, Mokl, shows that the protein is upregulated in myo52A cells. Localisation studies reveal that Mokl colocalises with Myo52 at the cell poles during tip growth and to a certain extent at the equator during cytokinesis. Mokl is delocalised in myo52A cells. These findings suggest that the function of Myo52 is to localise factors involved in growth, such as Mokl, to defined sites in the cell by virtue of its interaction with the actin cytoskeleton. CONTENTS Title Page ......................................................................................................1 Acknowledgements .......................................................................................2 Abstract.........................................................................................................3 Table of Contents ......................................................................................... 4 Figures and Tables .........................................................................................9 Abbreviations ................................................................................................11 A Note on Nomenclature .............................................................................. 13 Chapter 1 Introduction 1 THE CYTOSKELETON.................................................................. 17 1.1 Introduction ..................................................................................... 17 1.2 Actin ..................................................................................................17 1.3 Actin Binding Proteins ...................................................................... 19 1.4 Microtubules .....................................................................................20 1.5 Microtubule Associated Proteins ..................................................... 22 1.6 Intermediate Filaments ......................................................................22 1.2 MYOSINS........................................................................................ 26 1.2.1 The Myosin Superfamily ................................................................26 1.2.2 Myosin Structure ................................................ 29 a. Proteolytic Cleavage o f Myosin II..................................................29 b. Head Domain.................................................................................30 c. Neck Domain..................................................................................32 d. Tail Domain...................................................................................34 1.2.3 The Myosin Catalytic Cycle and Force Production ........................ 40 a. The Catalytic Cycle.........................................................................40 b. Force Production........................................................................... 41 1.2.4 Myosin Function ..............................................................................44 a. Class I Myosins..............................................................................44 b. Class II Myosins............................................................................ 47 c. Class V Myosins.............................................................................49 1.2.5 Myosins Participate in the Coordinated Transport of Cellular 52 Cargo Along Actin and Microtubule Tracks 1.3 FISSION YEAST..............................................................................55 1.3.1 Introduction .......................................................................................55 1.3.2 The Life Cycle of Schizosaccharomyces pombe...............................56 1.3.3 The Cell Division Cycle ....................................................................58 1.3.4 Growth and Septation in Fission Yeast ............................................ 60 1.3.5 The Fission Yeast Cytoskeleton .......................................................61 a. The Actin Cytoskeleton...................................................................61 b. The Microtubule Cytoskeleton........................................................64 1.3.6 Cell Morphogenesis in S. pombe.......................................................67 1,3.7 Cytokinesis in S. pombe.....................................................................70 a. Timing o f Cell Division.................................................................70 b. Cleavage Plane Specification........................................................ 72 c. Medial Ring Assembly.................................................................. 76 d. Medial Ring Contraction and Septation........................................81 1.4 OBJECTIVES OF THIS THESIS.....................................................89 Chapter 2 Materials and Methods 2.1 Identification and Analysis of Myosin Sequences ........................... 91 2.2 Chemicals............................................................................................91 2.3 Strains and Media ..............................................................................91 2.4 Plasmids ............................................................................................. 92 2.5 Oligonucleotides ................................................................................. 93 2.6 Preparation of Yeast Genomic DNA ..................................................94 2.7 Polymerase Chain Reaction (PCR) .....................................................95 2.8 Phenol:Chloroform Extraction of DNA ..............................................96 2.9 Ethanol Precipitation of DNA ............................................................96
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