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Tnik Are Novel Interaction Partners of Cnk2

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Tnik Are Novel Interaction Partners of Cnk2 Investigation of disease-associated scaffold proteins in the postsynaptic density Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Hanna Zieger Berlin, 2019 Die vorliegende Arbeit wurde im Zeitraum von 2014 bis 2018 im Neurowissenschaftlichen Forschungszentrum der Charité-Universitätsmedizin Berlin unter Anleitung von Professor Sarah Shoichet angefertigt. 1. Gutachterin: Professor Sarah Shoichet, NWFZ, Charité-Universitätsmedizin Berlin 2. Gutachterin: Professor Ursula Koch, Freie Universität Berlin Disputation am: 27. Mai 2019 Danksagung Ich danke meiner Mentorin Professor Sarah Shoichet dafür, dass ich in ihrem Labor an diesem spannenden Projekt arbeiten durfte. Besonderer Dank für ihr Verständnis, ihre Geduld, ihre Begeisterung und ihre stetige Unterstützung, die ich all die Jahre erfahren durfte. Stella danke ich für die wunderbare Betreuung, die vielen guten Gespräche und Diskussionen, die mir so oft weitergeholfen haben. Ebenso Nils, der immer Zeit und ein offenes Ohr hatte und nicht nur in fachlichen Fragen eine große Hilfe war. Bettina, für die Kollegialität und die interessanten Diskussionen, aber auch für ihre Unterstützung im Labor. Melanie, dafür dass sie sich um so vieles gekümmert hat und immer eine verlässliche Hilfe war. Ich hätte mir keine bessere Arbeitsgruppe wünschen können und jede/r hat auf seine/ihre Art dazu beigetragen, dass ich einen sehr lehrreiche, spannende, und tolle Zeit hatte. Ich danke der Advanced Medical Bioimaging Core Facility (AMBIO) der Charité sowie der Microscopy Core Facility (MCF) des NWFZ der Charité für ihre Unterstützung und die Bereitstellung der Mikroskope, sowie der Virus Core Facility (VCF) der Charité für die Produktion von Virus und die Bereitstellung von Laborraum. Besonderer Dank gilt Alexander Stumpf und Professor Dietmar Schmitz für die angenehme und spannende Kollaboration. Natürlich danke ich meinen Freunden fürs Dasein, Motivieren und Ablenken. Ich danke meiner großartigen Familie, auf die ich mich immer verlassen kann, und die durch Höhen und Tiefen an meiner Seite ist. Aus tiefstem Herzen danke ich Tom, der mich immer begleitet und wenn nötig auch ein Stück des Weges getragen hat; der mich immer wieder motiviert und in jeder Situation zum Lachen bringt. Content DANKSAGUNG ......................................................................................................................................................................... 3 INDEX OF FIGURES ................................................................................................................................................................... 1 INDEX OF TABLES ..................................................................................................................................................................... 2 DECLARATION ON THE PRESENTATION OF PUBLISHED DATA ................................................................................................. 3 SUMMARY ................................................................................................................................................................................ 4 ZUSAMMENFASSUNG .............................................................................................................................................................. 6 1. INTRODUCTION ............................................................................................................................................... 8 1.1 SYNAPSES – THE CONNECTIONS BETWEEN NEURONS ................................................................................................. 8 1.2 THE POSTSYNAPTIC DENSITY ........................................................................................................................................ 8 1.3 POSTSYNAPTIC SCAFFOLD PROTEINS ........................................................................................................................... 9 1.4 THE CNK SCAFFOLD PROTEIN FAMILY ..................................................................................................................... 11 1.4.1 The scaffold protein CNK is conserved ............................................................................................. 11 1.4.2 CNK2 in neurons ...................................................................................................................................... 13 1.4.3 CNK2 is disease associated ................................................................................................................... 14 2. MATERIALS AND METHODS ...................................................................................................................... 16 2.1 MATERIALS ................................................................................................................................................................ 16 2.1.1 Bacteria ....................................................................................................................................................... 16 2.1.2 Cell Culture ................................................................................................................................................ 16 2.1.3 Oligonucleotides ..................................................................................................................................... 17 2.1.4 Antibodies .................................................................................................................................................. 18 2.1.5 Reagents ..................................................................................................................................................... 19 2.1.6 Buffers and Solutions ............................................................................................................................. 21 2.1.7 Equipment ................................................................................................................................................. 22 2.2 METHODS .................................................................................................................................................................. 23 2.2.1 Expression Constructs ............................................................................................................................ 23 2.2.2 Co-immunoprecipitation, SDS-PAGE and western blot ............................................................. 24 2.2.3 Immunofluorescence COS-7 cells ...................................................................................................... 25 2.2.4 Neuron culture ......................................................................................................................................... 25 2.2.5 Lenti-viral infection and Immunofluorescence ............................................................................. 26 2.2.6 Imaging ....................................................................................................................................................... 26 2.2.7 Fluorescence Recovery After Photobleaching (FRAP) ................................................................ 26 2.2.8 Image Analysis .......................................................................................................................................... 29 2.2.9 Crude synaptosome preparation ....................................................................................................... 29 2.2.10 Custom-made CNK2 antibody ............................................................................................................ 30 2.2.11 Yeast-Two-Hybrid .................................................................................................................................... 30 3. RESULTS ......................................................................................................................................................... 31 3.1 CNK2 IS A MEMBRANE-ASSOCIATED PROTEIN EXPRESSED IN NEURONS AND ENRICHED AT POSTSYNAPTIC SITES 31 3.2 LOSS OF CNK2 INFLUENCES THE SIZE OF THE POSTSYNAPTIC DENSITY .................................................................. 33 3.3 EXPRESSION OF THE DISEASE-ASSOCIATED CNK2 VARIANT CAUSES INCREASED PSD SIZE AND INFLUENCES SYNAPTIC TRANSMISSION ..................................................................................................................................................... 35 3.3.1 CNK2 mutants ........................................................................................................................................... 36 3.3.2 PSD size is increased upon expression of disease-associated CNK2 ...................................... 38 3.3.3 mEPSC amplitude is increased in neurons expressing disease-associated CNK2 ............. 39 3.3.4 Mobility of the disease-associated CNK2 mutant is altered compared to the wild type 40 3.4 EXPRESSION OF A CNK2 VARIANT THAT DOES NOT BIND TO THE MEMBRANE AFFECTS PSD SIZE ......................... 42 3.5 THE KINASES MINK AND TNIK ARE NOVEL INTERACTION PARTNERS OF CNK2 .................................................... 45 3.5.1 Y2H screen to identify novel interaction partners ........................................................................ 45 3.5.2 CNK2 interacts with MINK1 and TNIK and regulates their localisation .................................
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