Role of FBXW5-loss in Centrosome Abnormalities and Cell Physiology Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Tim Scholta aus Spremberg Tübingen 2021 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 12.07.2021 Dekan: Prof. Dr. Thilo Stehle 1. Berichterstatter: Prof. Dr. Nisar Malek 2. Berichterstatter: Prof. Dr. Alfred Nordheim Content 1 Abbreviations ................................................................................................................................ 6 2 Abstract ......................................................................................................................................... 8 3 Zusammenfassung ....................................................................................................................... 9 4 Introduction ................................................................................................................................. 10 4.1 The importance of centrosomes in maintaining genomic stability ....................................... 10 4.2 Centrosome amplification – Cause or consequences of tumor formation? ......................... 12 4.3 Centrosome and Centrosome cycle ..................................................................................... 14 4.3.1 Disengagement/licensing of the centrosome ................................................................... 15 4.3.2 Initiation of centrosome duplication and maturation/elongation ...................................... 15 4.3.3 Centrosome disjunction and bipolar spindle formation .................................................... 17 4.4 The centrosome cycle initiation is controlled by the ubiquitin E3-ligase SCFFBXW5 ............. 17 4.5 Aim of the study ................................................................................................................... 18 5 Material and Methods ................................................................................................................. 20 5.1 Biologicals ............................................................................................................................ 20 5.1.1 Bacterial strains ............................................................................................................... 20 5.1.2 Cell lines used.................................................................................................................. 20 5.2 Medium and substances ...................................................................................................... 20 5.2.1 Antibiotics ......................................................................................................................... 20 5.2.2 Cell culture medium and substances ............................................................................... 20 5.2.3 Microbiological medium and substances ......................................................................... 21 5.2.4 Buffer and solutions for virus amplification ...................................................................... 22 5.2.5 Buffer and solutions for DNA-extraction, -purification and genotyping ............................ 22 5.2.6 Buffer and solutions for immunohistochemistry ............................................................... 23 5.2.7 Buffer and solutions for biochemical analysis .................................................................. 23 5.2.8 Oligonucleotides .............................................................................................................. 24 5.3 Methods................................................................................................................................ 28 5.3.1 Cultivation of bacteria ...................................................................................................... 28 5.3.2 E. coli-Transformation through heat shock ...................................................................... 28 5.3.3 Electroporation of E. coli .................................................................................................. 28 5.3.4 Cultivation and passaging of eukaryotic cells .................................................................. 29 5.3.5 Determination of doubling time of cells ............................................................................ 29 5.3.6 Migration assay ................................................................................................................ 30 5.3.7 Cryopreservation of cells ................................................................................................. 30 5.3.8 Transfection of BNL CL.2 cells ........................................................................................ 30 5.3.9 Virus work ........................................................................................................................ 31 5.3.10 Transduction of cells.................................................................................................... 32 5.3.11 Crystal violet staining................................................................................................... 32 5.3.12 Cell synchronization .................................................................................................... 32 5.3.13 Molecular biological methods ...................................................................................... 34 5.3.14 Plasmids used for the design of a conditional Fbxw5 targeting vector ....................... 36 5.3.15 Generation of CRISPR/Cas9 vectors, and microRNA-based shRNAs, to suppress FBXW5 expression in vitro and in vivo ......................................................................................... 38 5.3.16 The GeCKO CRISPR knockout library ........................................................................ 43 5.3.17 Isolation of Nucleic acids ............................................................................................. 45 5.3.18 cDNA synthesis ........................................................................................................... 47 5.3.19 Polymerase-Chain-Reaction (PCR) ............................................................................ 47 5.3.20 qPCR ........................................................................................................................... 48 5.3.21 Protein Isolation ........................................................................................................... 48 5.3.22 Discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ....................... 49 5.3.23 Western blot ................................................................................................................ 50 5.3.24 Histology methods ....................................................................................................... 51 5.3.25 Mouse work ................................................................................................................. 53 6 Results ......................................................................................................................................... 55 6.1 Validation of miR-shRNAs for FBXW5 suppression in vitro ................................................. 55 6.2 Validation of guide RNAs for CRISPR/Cas9 mediated-targeting of Fbxw5 in vitro ............. 56 6.3 Depletion of FBXW5 leads to abnormalities in centrosome number ................................... 57 6.4 Consequences of FBXW5 depletion for the regulation of G2M transition ............................ 60 6.5 Consequences of reduced FBXW5 expression for cell growth, migration and in vitro transformation ................................................................................................................................... 63 6.6 Creation of stable FBXW5 deficient cells for a genome wide screen using a CRISPR/Cas9 library ............................................................................................................................................. 65 6.7 CRISPR/Cas9 genome wide library identifies Replication Protein A2 (RPA2) to partially rescue FBXW5 knockout phenotype in BNL CL.2 cells .................................................................... 67 6.8 Analysis of the pathophysiological influence of FBXW5 suppression in an in vivo mosaic mouse model using the SB13 transposase system .......................................................................... 71 6.9 Creation of a transgenic floxed Fbxw5 gene for conditional Fbxw5 knockout models ........ 75 6.10 Insertion of the first loxP site at the 5’ UTR of Fbxw5 .......................................................... 77 6.11 Insertion of the FRT-NeoR-FRT-loxP site at the 3’ end of Fbxw5 ........................................ 79 6.12 Shuttling of floxed Fbxw5 into a eukaryotic expression vector for the generation of transgenic ES-cells ............................................................................................................................................. 81 7 Discussion ................................................................................................................................... 85 7.1 Loss of the Ubiquitin E3 Ligase FBXW5 impairs cell physiology ......................................... 85 7.2 A genome wide CRISPR/Cas9 library screen