Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Centrosome cohesion: functions of C-NAP1 Author(s) Flanagan, Anne-Marie Publication Date 2016-01-07 Item record http://hdl.handle.net/10379/5458 Downloaded 2021-09-25T13:41:15Z Some rights reserved. For more information, please see the item record link above. Centrosome Cohesion: Functions of C-NAP1 Anne-Marie Flanagan Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway A thesis submitted to the National University of Ireland, Galway for the degree of Doctor of Philosophy September 2015 Supervisor: Prof. Ciaran Morrison Table of Contents Table of Contents ....................................................................................ii List of figures ........................................................................................... v List of tables ......................................................................................... viii Abreviations ............................................................................................ ix Acknowledgements ................................................................................xii Abstract ................................................................................................ xiii 1. Introduction .................................................................................... 14 1.1 Cell cycle overview ..................................................................................... 15 1.2 DNA damage............................................................................................... 17 1.2.1 Single strand breaks ............................................................................. 17 1.2.2 Double strand breaks ............................................................................ 18 1.3 DNA break repair ...................................................................................... 19 1.3.1 Homologous recombination ................................................................. 19 1.3.2 Non-homologous end joining ............................................................... 20 1.4 DNA damage checkpoints ......................................................................... 21 1.4.1 G1 checkpoint ...................................................................................... 21 1.4.2 Intra S checkpoint ................................................................................ 22 1.4.3 G2/M checkpoint .................................................................................. 24 1.4.4 Spindle assembly checkpoint ............................................................... 24 1.5 The centrosome .......................................................................................... 25 1.6 Functions of the centrosome ..................................................................... 27 1.6.1 Microtubule nucleation ........................................................................ 27 1.6.2 Cell division ......................................................................................... 29 1.6.3 Cilia formation ..................................................................................... 32 1.6.4 Cellular signalling ................................................................................ 34 1.7 The centrosome cycle ................................................................................. 35 1.7.1 Overview of the centrosome cycle ....................................................... 35 1.7.2 Centriole disengagement ...................................................................... 37 1.7.3 Centrosome duplication ....................................................................... 38 1.8 Centrosome disjunction and the intercentriolar linker .......................... 40 1.9 Centriolar satellites .................................................................................... 43 1.9.1 Structure and cell cycle distribution ..................................................... 43 1.9.2 Centriolar satellites and ciliation .......................................................... 45 1.9.3 Anchoring and transportation functions of centriolar satellites ........... 46 ii 1.9.4 Centriolar satellites and the cell cycle .................................................. 47 1.10 Centrosome amplification ......................................................................... 49 1.10.1 Tetraploidy and Polyploidy .................................................................. 49 1.10.2 Centrosome protein overexpression ..................................................... 50 1.10.3 De novo centriole formation................................................................. 51 1.11 DNA damage and centrosome amplification ........................................... 53 1.12 Centrosomes and cancer ............................................................................ 54 1.13 Aims of this study, model systems and targeting methods ..................... 57 2. Materials and methods .................................................................. 58 2.1 Reagents and materials.............................................................................. 59 2.1.1 Materials ............................................................................................... 59 2.1.2 Biological materials ............................................................................. 65 2.2 Bacterial culture methods ......................................................................... 65 2.2.1 Preparation of E. coli for transformation ............................................. 65 2.2.2 Transformation of chemically competent E. coli ................................. 65 2.3 Nucleic acid methods ................................................................................. 66 2.3.1 RNA isolation and cDNA synthesis ..................................................... 66 2.3.2 Extraction of genomic DNA ................................................................ 66 2.3.3 Polymerase chain reaction.................................................................... 67 2.3.4 DNA cloning methods ......................................................................... 67 2.3.5 Assembly of target CRISPR plasmids ................................................. 69 2.3.6 Analysis of cloned DNA and sequencing ............................................ 69 2.3.7 DIG labelling of DNA probes .............................................................. 70 2.3.8 Southern blot ........................................................................................ 70 2.4 Protein Methods ......................................................................................... 71 2.4.1 Extraction of protein from cells ........................................................... 71 2.4.2 Protein quantification ........................................................................... 71 2.4.3 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..................... 71 2.4.4 Western Blot......................................................................................... 72 2.5 Cell biology methods .................................................................................. 73 2.5.1 Cell maintenance .................................................................................. 73 2.5.2 Transient transfection ........................................................................... 74 2.5.3 Stable cell line generation .................................................................... 75 2.5.4 Auxin mediated protein depletion ........................................................ 76 2.5.5 Microtubule regrowth assay ................................................................. 76 2.5.6 Serum starvation................................................................................... 76 2.5.7 Flow cytometry .................................................................................... 76 iii 2.5.8 Microscopy methods ............................................................................ 77 2.5.9 Computer programmes ......................................................................... 78 3. Results: Reverse genetic analysis of C-NAP1 in the DT40 cell line................................................................................................... 80 3.1 Identification of the Gallus gallus C-NAP1 locus .................................... 81 3.2 Analysis of the chicken C-NAP1 transcript ............................................. 84 3.3 C-NAP1 localisation and function in DT40 cells ..................................... 87 3.3.1 Generation of C-NAP1-AIDGFP cell line ........................................... 87 3.3.2 Verification of C-NAP1-AIDGFP cell line .......................................... 88 3.3.3 Centrosome cohesion in C-NAP1off cells ............................................. 93 3.3.4 Centrosomal responses to DNA damage after C-NAP1 depletion ...... 94 4. Results: Disruption of human C-NAP1 ........................................ 97 4.1 Analysis of the HsC-NAP1 locus ............................................................... 98 4.2 Cloning and overexpression of C-NAP1 .................................................. 99 4.3 Novel C-NAP1 antibody and RNA interference of C-NAP1 ................ 100 4.4 C-NAP1 detection and overexpression in hTERT-RPE1 cells ............ 102 4.5 Targeting C-NAP1 using