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Drosophila Melanogaster Impact of Baz/PAR-3 phosphorylation by aPKC on cell polarity DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR.RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Sabine Ursula Katharina Feicht aus Burglengenfeld im Jahr 2017 Das Promotionsgesuch wurde eingereicht am: 10.03.2017 Die Arbeit wurde angeleitet von: Junior Prof. Dr. med. vet. Dr. rer. nat. Michael Krahn Unterschrift: Table of contents 1 Zusammenfassung ................................................................................................ 6 2 Summary ............................................................................................................... 7 3 Introduction .......................................................................................................... 8 3.1 Cell polarity ....................................................................................................... 8 3.2 Epithelial cell polarity in the model organisms Drosophila melanogaster ....... 9 3.3 The PAR complex ............................................................................................ 13 3.4 Bazooka/PAR-3 ............................................................................................... 16 3.5 The serine/theronine kinase aPKC ................................................................... 18 3.6 The adaptor protein PAR-6 .............................................................................. 19 3.7 The Crb complex ............................................................................................. 20 3.8 The embryonic development of Drosophila melanogaster ............................. 21 3.9 Research objectives .......................................................................................... 23 4 Material & Methods ........................................................................................... 24 4.1 Material ............................................................................................................ 24 4.1.1 Reagents ............................................................................................... 24 4.1.2 Solutions and buffer ............................................................................. 26 4.1.3 Media and agarose plates ..................................................................... 29 4.1.4 Oligonucleotides .................................................................................. 31 4.1.5 Plasmids ............................................................................................... 34 4.1.6 Commercial kits ................................................................................... 34 4.1.7 Antibodies ............................................................................................ 35 4.1.8 Enzymes ............................................................................................... 37 4.1.9 Marker .................................................................................................. 39 4.1.10 Fly lines ................................................................................................ 39 4.1.11 Cell lines .............................................................................................. 40 4.1.12 Bacterial strains .................................................................................... 41 4.1.13 Disposables .......................................................................................... 41 4.1.14 Devices ................................................................................................. 42 4.1.15 Data bases and software ....................................................................... 44 4.2 Methods ........................................................................................................... 45 4.2.1 Molecular biology methods ................................................................. 45 4.2.1.1 Polymerase chain reaction (PCR) ................................................. 45 4.2.1.1.1 Mutagenesis PCR ...................................................................... 46 4.2.1.2 Agarose gel electrophoresis .......................................................... 47 4.2.1.3 Molecular cloning ......................................................................... 47 4.2.1.4 Subcloning via Entry/GatewayTM technology ............................ 48 4.2.1.5 Transformation of chemical competent E. coli cells .................... 50 4.2.1.6 Isolation of plasmid DNA via alkaline lysis ................................. 50 4.2.1.6.1 Mini Preparation ........................................................................ 50 4.2.1.6.2 Midi Preparation ........................................................................ 51 4.2.1.7 Analysis of plasmid DNA via analytical digestion ...................... 52 4.2.2 Biochemical methods ........................................................................... 52 4.2.2.1 Protein extraction from Drosophila embryos ............................... 52 4.2.2.2 Protein extraction from Drosophila Schneider cells .................... 53 4.2.2.3 Measurement of protein concentration ......................................... 53 4.2.2.4 Co-immunoprecipitation ............................................................... 54 4.2.2.5 SDS-PAGE ................................................................................... 54 4.2.2.6 Western blot .................................................................................. 55 4.2.2.7 Protein purification ....................................................................... 56 4.2.2.8 In vitro kinase assay ..................................................................... 57 4.2.3 Drosophila cell culture ........................................................................ 58 4.2.3.1 Transfection with FuGene®HD Transfection Reagent ................. 58 4.2.4 Fly genetics .......................................................................................... 59 4.2.4.1 Drosophila melanogaster breeding conditions ............................ 59 4.2.4.2 The PhiC31 integrase system ....................................................... 60 4.2.4.3 The dominant female sterile technique ......................................... 61 4.2.4.4 The UAS-GAL4 system ............................................................... 63 4.2.4.5 Lethality test ................................................................................. 64 4.2.5 Histology .............................................................................................. 64 4.2.5.1 Fixation and immunostaining of Drosophila embryos ................. 64 4.2.5.2 Detection of apoptosis in Drosophila embryos ............................ 65 4.2.5.3 Confocal microscopy .................................................................... 66 4.2.5.4 Cuticle preparation of Drosophila embryos ................................. 66 5 Results ................................................................................................................ 67 5.1 Localization of aPKC is shifted in early development of Drosophila embryonic epithelia ................................................................................................. 67 5.2 Baz PDZ domains and aPKC-binding region are involved in aPKC binding . 68 5.3 Baz/PAR-3PDZ2-3 domain is phosphorylated by aPKC at multiple residues .... 70 5.4 The impact of aPKC-mediated phosphorylation of BazPDZ2-3 in vivo .............. 75 5.5 Phosphorylation of BazPDZ2-3 is important for aPKC binding and protein stability .................................................................................................................... 77 5.6 Phosphorylation of BazPDZ2-3 by aPKC is important for early Drosophila embryonic development .......................................................................................... 79 5.7 aPKC mediated phosphorylation of BazPDZ2-3 is crucial for early Drosophila embryonic development due to a lack of Crb expression ....................................... 86 5.8 Phosphorylation of BazPDZ2-3 by aPKC is important to restrict basolateral PAR-1 activity ......................................................................................................... 88 5.9 The impact of kinase activities on the phosphorylation of BazPDZ2-3 .............. 90 5.10 The impact of PAR-6 on the kinase activity of aPKC ..................................... 93 6 Discussion ........................................................................................................... 99 6.1 Structural and functional analysis of the BazPDZ2-3 phosphorylation by aPKC100 6.2 The relevance of BazPDZ2-3 phosphorylation in early epithelial development104 6.3 The impact of kinase activity on the establishment of early epithelial polarity107 7 References ........................................................................................................ 110 8 Appendix .......................................................................................................... 119 8.1 List of Figures ................................................................................................ 119 8.2 List of Tables ................................................................................................. 120 8.3 Abbreviations ................................................................................................. 121 9 Acknowledgements .........................................................................................
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