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Investigating the Actin Regulatory Activities of Las17, the Wasp Homologue in S. Cerevisiae Liemya E. Abugharsa

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Investigating the Actin Regulatory Activities of Las17, the Wasp Homologue in S. Cerevisiae Liemya E. Abugharsa Investigating the actin regulatory activities of Las17, the WASp homologue in S. cerevisiae A thesis submitted for the degree of Doctor of Philosophy By Liemya E. Abugharsa Department of Molecular Biology and Biotechnology University of Sheffield March 2015 Abstract Investigating the actin regulatory activities of Las17, the WASp homologue in S. cerevisiae Clathrin mediated endocytosis (CME) in S. cerevisiae requires the dynamic interplay between many proteins at the plasma membrane. Actin polymerisation provides force to drive membrane invagination and vesicle scission. The WASp homologue in yeast, Las17 plays a major role in stimulating actin filament assembly during endocytosis. The actin nucleation ability of WASP family members is attributed to their WCA domain [WH2 (WASP homology2) domain, C central, and A (acidic) domains] which provides binding sites for both actin monomers and the Arp2/3 complex. In addition, the central poly-proline repeat region of Las17 is able to bind and nucleate actin filaments independently of the Arp2/3 complex. While Las17 is a key regulator of endocytic progression and has been found to be phosphorylated in global studies, the mechanism behind regulation of Las17 actin-based function is unclear. Therefore, the aims of this study were to investigate the post-translation modification of Las17 by phosphorylation, and to determine how this modification impacts on Las17 function both in vivo and in vitro. Mass Spec analysis was employed and allowed identification of further phosphorylation sites in Las17. Through the studies described here I was able to demonstrate that Las17 is phosphorylated, and that one specific phosphorylation event was of importance in endocytosis. Ser588 mutants were shown to affect an early stage of endocytosis whereby S588D mutant had a negative impact on growth, actin organisation, and endocytic invagination. The overall results suggest a regulatory model in which Las17 is subjected to intramolecular interaction that supports Arp2/3- independent actin nucleation mediated by its PP region. Phosphorylation must then be relieved to allow Arp2/3-dependent actin polymerisation to take place to drive invagination. These data establish a novel regulatory model of Las17 and have increased our understanding on the mechanism of actin nucleation during early endocytosis in yeast. i Table of contents Abstract ...................................................................................................................i Table of contents ................................................................................................. ii List of figures .................................................................................................... viii List of tables ....................................................................................................... x Abbreviations .........................................................................................................xi Chapter 1: Introduction 1.1 General introduction ......................................................................................... 1 1.2 Actin ................................................................................................................ 4 1.2.1 Actin structures ............................................................................................ 4 1.2.1.1 Monomeric (G)-actin .................................................................................. 4 1.2.1.2 The actin polymer (F-actin) ........................................................................ 8 1.2.2 Actin polymerisation ...................................................................................... 9 1.2.2.1 Nucleation ................................................................................................ 11 1.2.2.2 Elongation ................................................................................................ 13 1.2.2.3 Treadmiling .............................................................................................. 15 1.3 Cytoskeleton in prokaryotic ............................................................................ 15 1.4 Actin binding proteins (ABPs) ........................................................................ 18 1.4.1 G-actin binding proteins .............................................................................. 20 1.4.1.1 Profilin family ........................................................................................... 20 1.4.1.2Thymosin β4 ............................................................................................. 21 1.4.1.3 Twinfilin .................................................................................................... 21 1.4.2 F-actin binding proteins ............................................................................... 22 1.4.2.1 Capping -CapZ ......................................................................................... 22 1.4.2.2 F-actin depolymerisation proteins- ADF/Cofilin......................................... 22 1.4.2.3 F-actin severing proteins .......................................................................... 23 1.4.3 Actin crosslinking/bundling proteins ............................................................ 25 1.4.4 Myosins ...................................................................................................... 26 1.4.5 Actin stabilizers ........................................................................................... 26 1.4.6 Actin nucleators .......................................................................................... 27 1.4.6.1 Arp2/3 Complex ....................................................................................... 27 1.4.6.2 Formins .................................................................................................... 31 1.4.7 Nucleation promoting factors (NPFs) .......................................................... 33 1.4.7.1 NPFs class I ............................................................................................ 33 1.4.7.1.1WASP and N-WASP .............................................................................. 33 1.4.7.1.1.1Regulation of WASPs by phosphorylation ........................................... 36 1.4.7.1.2 Ena/VASP ............................................................................................. 39 1.4.7.1.3 WAVE/SCAR ........................................................................................ 39 1.4.7.1.4 WASH ................................................................................................... 40 1.4.7.1.5 WHAMM ............................................................................................... 41 1.4.7.1.6 JMY ...................................................................................................... 42 1.4.7.2 NPFs class II (Cortactin) .......................................................................... 42 ii Table of contents 1.5 Actin in yeast ................................................................................................. 44 1.5.1 Actin architecture in yeast ........................................................................... 45 1.5.1.1 Actin cortical patches ............................................................................... 45 1.5.1.2 Actin cables ............................................................................................. 45 1.5.1.3 Acto-myosin ring ...................................................................................... 46 1.6 Role of actin in yeast ..................................................................................... 48 1.7 Endocytosis in yeast ...................................................................................... 49 1.7.1 Actin binding proteins(ABPs) in yeast ......................................................... 50 1.7.2 Stages of yeast endocytosis ....................................................................... 50 1.7.2.1 Stage1: non-motile ................................................................................... 53 1.7.2.2 Stage 2: slow movement (invagination) .................................................... 55 1.7.2.2.1 Regulation of actin polymerisation in endocytosis ................................ 56 1.7.2.3 Stage 3: vesicle scission and uncoating .................................................. 59 1.8 The WASp homologue, Las17 ....................................................................... 61 1.9 Roles of Las17 in endocytosis ...................................................................... 66 1.10 Summary ..................................................................................................... 67 1.11 Aims of the project ....................................................................................... 68 Chapter 2: Materials and Methods 2.1 Materials ........................................................................................................ 69 2.2 Yeast strains, plasmids, oligonucleotides, and antibodies ............................. 96 2.3 Molecular Biology Techniques ...................................................................... 73 2.3.1 DNA plasmid mini prep .............................................................................. 73 2.3.2 DNA Agarose Gel Electrophoresis ............................................................ .73 2.3.3 DNA Extraction from Agarose Gel .............................................................
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