Functional analysis of STRIPAK complex components in the filamentous ascomycete Sordaria macrospora Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Microbiology and Biochemistry of the Georg-August University School of Science (GAUSS) submitted by Eva Johanna Reschka from Tarnowitz (Poland) Göttingen, 2017 Members of the Thesis Committee Prof. Stefanie Pöggeler Department of Genetics of Eukaryotic Microorganisms, Institute of Microbiology and Genetics Prof. Gerhard Braus Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics Prof. Henning Urlaub Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry Members of the Examination Board 1st Referee Prof. Stefanie Pöggeler Department of Genetics of Eukaryotic Microorganisms, Institute of Microbiology and Genetics 2nd Referee Prof. Kai Heimel Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics Further members of the Examination Board Prof. Gerhard Braus Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics Prof. Henning Urlaub Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry Prof. Rolf Daniel Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics Prof. Heike Krebber Department of Molecular Genetics, Institute of Microbiology and Genetics Date of oral examination: 18.10.2017 Affirmation I hereby declare that this thesis was written independently and with no other sources and aids than quoted. Göttingen, 25.08.2017 Eva Johanna Reschka This doctoral study was performed in the group of Prof. Stefanie Pöggeler in the Department of Genetics of Eukaryotic Microorganisms at the Institute of Microbiology and Genetics, Georg- August University Göttingen. Table of Contents I. Table of Contents I. Table of Contents ......................................................................................................... i II. List of figures ........................................................................................................... viii III. List of tables ............................................................................................................... x IV. List of figures in the Supplement ................................................................................ xi V. List of videos in the Supplement/DVD ....................................................................... xii VI. List of Abbreviations ................................................................................................ xiii Summary ......................................................................................................................... 1 Zusammenfassung .......................................................................................................... 2 1. Introduction ............................................................................................................... 4 1.1 Components of mammalian STRIPAK complex ............................................................ 6 1.1.1 Striatins .................................................................................................................... 6 1.1.2 PP2A ......................................................................................................................... 8 1.1.3 STRIP1/2 ................................................................................................................... 9 1.1.4 SLMAP/TRAF3IPS ................................................................................................... 10 1.1.5 CTTNBP2/CTTNBP2NL ............................................................................................ 11 1.1.6 MOB3 (phocein) ..................................................................................................... 12 1.1.7 Germinal center kinases and CCM3....................................................................... 13 1.1.7.1 MST3 ........................................................................................................ 14 1.1.7.2 MST4 and STK25 ...................................................................................... 14 1.1.7.3 MINK ........................................................................................................ 14 1.1.8 SIKE/FGFR1OP2 ...................................................................................................... 15 i Table of Contents 1.1.8.1 SIKE ........................................................................................................... 15 1.1.8.2 FGFR1OP2 ................................................................................................ 15 1.2 STRIPAK-like complexes in yeasts and in Drosophila melanogaster ........................... 16 1.2.1 The SIP-SIN-MOR crosstalk in S. pombe ................................................................. 16 1.2.2 The Far complex in Saccharomyces cerevisiae ...................................................... 18 1.2.2.1 The Far complex in S. cerevisiae is involved in pheromone-mediated cell cycle arrest ............................................................................................... 18 1.2.2.2 The Far complex regulates cell growth, autophagy and actin polarization .................................................................................................................. 20 1.2.2.3 MEN regulates mitotic exit and cytokinesis in S. cerevisiae .................... 21 1.2.3 STRIPAK complex in Drosophila melanogaster ...................................................... 22 1.2.3.1 Hippo signaling in D. melanogaster ......................................................... 22 1.2.3.2 Cka in the Jun-N-terminal kinase pathway in D. melanogaster .............. 24 1.2.3.3 The dSTRIPAK activates the Ras-Raf-MAPK/ERK pathway ...................... 25 1.3 Sordaria macrospora is a model organism to study fruiting-body development ......... 26 1.3.1 The STRIPAK complex in S. macrospora regulates sexual development and hyphal fusion .......................................................................................................... 27 1.3.2 Striatin orthologs in filamentous fungi control hyphal fusion, sexual development and vegetative growth ..................................................................... 28 1.3.3 Fungal MOB3 proteins are involved in hyphal fusion, development and vesicular traffic ...................................................................................................................... 29 1.3.4 The fungal STRIP1/2 ortholog and the catalytic PP2ac1 subunit link the fungal STRIPAK to septation and the CWI pathway .......................................................... 30 1.3.5 The SLMAP ortholog PRO45 is part of the fungal STRIPAK complex in S. macrospora ............................................................................................................ 31 1.3.6 Fungal STRIPAK-associated proteins SmGPI1 and GCKIII kinases SmKIN3 and SmKIN24 ................................................................................................................. 31 1.4 Aims of this thesis..................................................................................................... 32 2. Materials and Methods ............................................................................................ 34 ii Table of Contents 2.1 Materials .................................................................................................................. 34 2.1.1 Strains .................................................................................................................... 34 2.1.2 Plasmids ................................................................................................................. 38 2.1.3 Primers ................................................................................................................... 41 2.1.4 Chemicals and materials ........................................................................................ 46 2.1.4.1 Chemicals ................................................................................................. 46 2.1.4.2 Materials .................................................................................................. 47 2.1.5 Kits ......................................................................................................................... 47 2.1.6 Enzymes ................................................................................................................. 48 2.1.7 Antibodies .............................................................................................................. 48 2.1.8 Buffers and solutions ............................................................................................. 48 2.1.9 Media ..................................................................................................................... 51 2.2 Methods ................................................................................................................... 52 2.2.1 Cultivation of microorganisms ............................................................................... 52 2.2.2 Preparation of competent microorganisms and transformation procedure ........ 53 2.2.3 Generation of S. macrospora deletion strains ....................................................... 54 2.2.3.1 Generation of S. macrospora single