Function and Regulation of Kinesin-1, -2 and -3 A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2010 Kim Diane Brownhill Table of Contents List of Figures................................................................................ 6 List of Tables ................................................................................. 8 Abstract .......................................................................................... 9 Declaration ................................................................................... 10 Copyright statement.................................................................... 11 Acknowledgments ....................................................................... 12 List of abbreviations ................................................................... 13 1. Introduction ............................................................................. 15 1.1 Cytoskeleton ..................................................................................................................... 16 1.1.1 Actin and intermediate filaments ............................................................................... 16 1.1.2 Microtubules .............................................................................................................. 17 1.2 Microtubule motors ........................................................................................................... 20 1.2.1 Kinesin-1 .................................................................................................................... 20 1.2.2 Kinesin-2 .................................................................................................................... 21 1.2.3 Kinesin-3 .................................................................................................................... 23 1.2.4 Cytoplasmic dynein-1 ................................................................................................ 25 1.3 Motor regulation ................................................................................................................ 26 1.3.1 Motor phosphorylation ............................................................................................... 26 1.3.2 Calcium signalling ..................................................................................................... 27 1.3.3 Autoinhibition of kinesin-1 ......................................................................................... 28 1.3.4 Rab GTPases ............................................................................................................ 29 1.3.5 Microtubule modifications .......................................................................................... 30 1.4 Transport in the secretory and endocytic pathways ......................................................... 31 1.4.1 ER-to-Golgi traffic ...................................................................................................... 31 1.4.2 Golgi-to-ER traffic ...................................................................................................... 32 1.4.2.1 COPI-dependent and –independent pathways .................................................. 32 1.4.2.2 BFA .................................................................................................................... 32 1.4.3 Post-Golgi trafficking ................................................................................................. 35 1.4.4 Endocytosis ............................................................................................................... 36 1.5 Organelle dynamics in mitosis .......................................................................................... 38 1.5.1 Cell cycle ................................................................................................................... 38 1.5.2 ER motility and inheritance ........................................................................................ 39 1.5.3 Golgi inheritance and trafficking processes .............................................................. 43 1.5.4 Xenopus laevis egg extracts ..................................................................................... 44 1.6 Aims of the studies ........................................................................................................... 46 1.6.1 Cell cycle regulation of kinesin-1 ............................................................................... 46 1.6.2 Motility of BFA-induced tubules ................................................................................. 47 2 2. Materials and Methods ............................................................ 54 2.1 Solutions/reagents ............................................................................................................ 55 2.2 Cell-based assays ............................................................................................................ 57 2.2.1 Cell culture ................................................................................................................. 57 2.2.2 Transient transfection ................................................................................................ 58 2.2.3 KIF1C siRNA ............................................................................................................. 58 2.2.4 BFA treatment ........................................................................................................... 59 2.2.5 Immunofluorescence ................................................................................................. 60 2.2.5.1 Methanol fixation ................................................................................................ 60 2.2.5.2 PFA fixation ........................................................................................................ 60 2.2.5.3 PFA/Glutaraldehyde fixation .............................................................................. 60 2.2.5.4 Antibody labelling ............................................................................................... 60 2.2.5.5 Imaging .............................................................................................................. 60 2.3 Antibodies ......................................................................................................................... 61 2.3.1 Primary antibodies ..................................................................................................... 61 2.3.2 Secondary antibodies ................................................................................................ 62 2.4 Protein analysis ................................................................................................................ 62 2.4.1 SDS-PAGE ................................................................................................................ 62 2.4.2 Coomassie stain ........................................................................................................ 62 2.4.3 Colloidal Coomassie stain ......................................................................................... 62 2.4.4 Western blot .............................................................................................................. 63 2.4.5 IP ............................................................................................................................... 63 2.5 Molecular biology .............................................................................................................. 63 2.5.1 Constructs ................................................................................................................. 63 2.5.2 Primers ...................................................................................................................... 64 2.5.3 Polymerase chain reaction (PCR) ............................................................................. 64 2.5.4 DNA digestion............................................................................................................ 64 2.5.5 DNA gel extraction .................................................................................................... 65 2.5.6 Ligation ...................................................................................................................... 65 2.5.7 Transformation of ligation .......................................................................................... 65 2.5.8 DNA Sequencing ....................................................................................................... 65 2.5.9 Constructs generated ................................................................................................ 66 2.5.9.1 pGex-KG-KBPΔ250-81 ...................................................................................... 66 2.5.9.2 pGex-KG-E-MAP-115 ........................................................................................ 66 2.6 Recombinant protein expression ...................................................................................... 67 2.6.1 Protein expression and purification ........................................................................... 67 2.6.2 GST-KBPΔ250-81 ..................................................................................................... 67 2.6.3 K430-BCCP ............................................................................................................... 67 2.7 Xenopus laevis