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Dynamics of the Linc Complex

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Dynamics of the Linc Complex DYNAMICS OF THE LINC COMPLEX Loic Gazquez University of Manchester School of Biological Sciences 2017 A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health TABLE OF CONTENTS Table of Contents .................................................................................................................................... 2 Abstract.................................................................................................................................................... 4 Declaration .............................................................................................................................................. 5 Copyright statement ................................................................................................................................ 5 Acknowledgements ................................................................................................................................. 6 I. Introduction .................................................................................................................................. 7 Nuclear Envelope and the LINC complex ................................................................................... 7 I. 1.1. The first LINC component: the SUN ................................................................................ 8 I. 1.2. The second LINC component: the KASH ........................................................................ 8 I. 1.1. Structure and function of the LINC complex .................................................................... 9 I. 1.1. Nuclear envelopathies and dystonia.............................................................................. 11 TorsinA ...................................................................................................................................... 12 I. 2.1. Alterations to the activity of TorsinA .............................................................................. 13 I. 2.2. Function and binding partners of TorsinA ..................................................................... 14 I. 2.3. Evidence for a role of TorsinA in the Nuclear envelope ................................................ 17 Nuclear envelope breakdown during mitosis ............................................................................. 18 Aims and objectives ................................................................................................................... 20 II. Materials and methods .............................................................................................................. 21 Methods ............................................................................................................................. 21 II. 1.1. Nucleic acid procedures ................................................................................................ 21 II. 1.2. Tissue culture procedures ............................................................................................. 27 II. 1.3. Protein procedures ........................................................................................................ 29 II. 1.4. Fluorescence microscopy .............................................................................................. 36 II. 1.5. Quantification of immunofluorescence images .............................................................. 37 Materials ............................................................................................................................ 39 III. The LINC Complex during mitosis ............................................................................................. 42 Introduction ........................................................................................................................ 42 Overlap between endogenous LINC components during cell division .............................. 42 III. 2.1. SUN1 and Nup153 have different distributions during mitosis .................................. 43 III. 2.1. SUN1 and SUN2 mostly overlap but distribute differently during telophase ............. 43 III. 2.2. SUN1 and nesprin-2 overlap during the whole mitotic cycle ..................................... 45 III. 2.3. SUN1 and nesprin-2 but not SUN2 overlap in clusters during telophase ................. 48 Nesprin-2 does not have the exact same timing as neither SUN2 nor SUN1 in returning to the NE 50 III. 3.1. Developing cell lines expressing GFP-nesprin-2 ....................................................... 50 III. 3.2. SUN1 and GFP-nesprin-2 overlap in telophase clusters free of SUN2..................... 52 III. 3.3. SUN1 leaves the cleavage furrow sooner than GFP-nesprin-2 and SUN2............... 53 III. 3.4. Clusters of SUN1/nesprin-2 are located in the ER .................................................... 54 III. 3.5. SUN1 and GFP-nesprin-2 clusters persist in early G1 .............................................. 54 Effect of TorsinA depletion on the LINC complex during cell division ............................... 55 III. 4.1. Optimisation for TorsinA knock-down ........................................................................ 56 III. 4.2. GFP-nesprin-2 during the mitotic cycle when TorsinA is reduced ............................ 58 Effect of the exogenous LAP1-V5-BioID on the LINC complex during cell division .......... 59 2 | P a g e III. 5.1. Overexpressing LAP1-V5-BioID causes an increase in the numbers of GFP-nesprin-2 clusters Error! Bookmark not defined. Discussion ......................................................................................................................... 62 IV. LINC complex disassembly and degradation ............................................................................ 66 Introduction ........................................................................................................................ 66 Nesprin-2 is subjected to proteasomal degradation .......................................................... 66 Displacement of GFP-nesprin-2 from the NE .................................................................... 70 IV. 3.1. KASH4 recombinant protein does not affect GFP-nesprin-2 .................................... 71 IV. 3.1. SUN1 knock-down decreases GFP-nesprin-2 levels ................................................ 72 IV. 3.2. ER-localised SUN1 recombinant protein displaces GFP-nesprin-2 to the ER and decreases GFP-nesprin-2 levels ................................................................................................... 79 Displacement of nesprin-2 from the NE............................................................................. 85 IV. 4.1. SUN1 knock-down does not affect endogenous nesprin-2 levels ............................. 85 IV. 4.2. KASH4 recombinant protein displaces nesprin-2 to the ER and increases nesprin-2 levels 87 IV. 4.3. ER-localised soluble SUN1 recombinant protein displaces nesprin-2 to the ER and increases nesprin-2 levels ............................................................................................................. 89 Effect of TorsinA and LAP1 knock-down on the LINC complex ........................................ 91 IV. 5.1. Optimisation of LAP1 knock-down............................................................................. 91 IV. 5.2. Disrupting TorsinA decreases the levels of GFP-nesprin-2 ...................................... 92 IV. 5.3. TorsinA knock-down increases the levels of endogenous LINC components .......... 94 IV. 5.4. TorsinA knock-down increases the mobility of GFP-nesprin-2 ................................. 97 Dicussion ........................................................................................................................... 98 V. Interactome network with proximity labelling ........................................................................... 103 Introduction ...................................................................................................................... 103 Generation of cells stably expressing the baits ............................................................... 103 Characterisation of the BioID biotin ligase ...................................................................... 107 Pull-down and biotinylation optimisation ......................................................................... 110 Analysis of the pull-downs ............................................................................................... 115 Discussion ....................................................................................................................... 127 VI. General discussion and future work ........................................................................................ 131 References .......................................................................................................................................... 134 Word count: 47,583 3 | P a g e ABSTRACT Name of the University: The University of Manchester Candidate’s full name: Loic Gazquez Degree Title: PhD in Medicine Thesis Title: Dynamics of the LINC Complex Date: 2017 The LINC complex is composed of SUN and KASH domain proteins spanning the nuclear envelope (NE), allowing communication between the cytoskeleton and the nucleoskeleton. Mutations in these proteins can result in diseases known as nuclear envelopathies. Early Onset Torsion Dystonia (EOTD) is caused by a mutation in a protein called TorsinA
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