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Rho Gtpases and Their Regulators in Cell Polarity of the Filamentous Ascomycete Neurospora Crassa

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Rho Gtpases and Their Regulators in Cell Polarity of the Filamentous Ascomycete Neurospora Crassa Rho GTPases and their regulators in cell polarity of the filamentous ascomycete Neurospora crassa Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades "Doctor rerum naturalium" der Georg-August-Universität Göttingen vorgelegt von Corinna Richthammer aus Nürnberg Göttingen 2011 Mitglied des Betreuungsausschusses: Dr. Stephan Seiler (Referent) Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen Mitglied des Betreuungsausschusses: Prof. Dr. Stefanie Pöggeler (Referentin) Abteilung Genetik eukaryotischer Mikroorganismen, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen Mitglied des Betreuungsausschusses: Prof. Dr. Andreas Wodarz Abteilung Stammzellbiologie, Göttinger Zentrum für Molekulare Biowissenschaften, Georg- August-Universität Göttingen Tag der mündlichen Prüfung: I hereby confirm that this thesis has been written independently and with no other sources and aids than quoted. Göttingen, 31.01.2011 Corinna Richthammer Parts of this work have been or will be published in: Justa-Schuch, D., Heilig, Y., Richthammer, C., and Seiler, S. (2010). Septum formation is regulated by the RHO4-specific exchange factors BUD3 and RGF3 and by the landmark protein BUD4 in Neurospora crassa. Mol. Microbiol 76, 220-235. Riquelme, M., Yarden, O., Bartnicki-Garcia, S., Bowman, B., Castro-Longoria, E., F ee J F ei e F ei g e i -Pérez, R., Plamann, M., Rasmussen, C., Richthammer, C., Roberson, R. W, Sanchez-Leon, E., Seiler, S., and Watters, M. K. Architecture and development of the Neurospora crassa hypha – a model cell for polarized growth. (Article in press, to appear in Fungal Biol., 2011) Table of Contents i Table of Contents 1. Summary ............................................................................................................................ 1 2. Zusammenfassung ........................................................................................................... 3 3. Introduction ....................................................................................................................... 5 3.1 Structure and mechanism of Rho GTPases ............................................................... 5 3.2 General regulation of Rho GTPases ........................................................................... 6 3.3 Effector pathways, cellular functions and fine-tuning of Rho GTPase signalling ....... 8 3.4 Rho GTPases and polarized growth in yeasts ............................................................ 9 3.4.1 Rho GTPases in yeasts: An overview ................................................................. 9 3.4.2 Polarized growth and its characteristics in yeasts ............................................. 10 3.4.3 The Cdc42 GTPase module and its role in establishment of polarity ............... 11 3.4.3.1 The Cdc42p module in S. cerevisiae .......................................................... 11 3.4.3.2 The Cdc42 module in S. pombe ................................................................. 12 3.4.4 The Rho1 GTPase module as a guardian of cell wall integrity ......................... 13 3.4.4.1 The Rho1p module in S. cerevisiae ............................................................ 13 3.4.4.2 The Rho1 module in S. pombe ................................................................... 16 3.5 Rho GTPases and polarized growth in filamentous fungi ......................................... 18 3.5.1 Characteristics of hyphal growth in filamentous fungi ....................................... 18 3.5.2 The roles of Rac and Cdc42 homologues in filamentous fungi ......................... 20 3.5.3 The functions of Rho1 homologues in filamentous fungi................................... 23 3.6 Rho GTPases and their regulators in N. crassa ....................................................... 25 3.7 Aims of this work ........................................................................................................ 26 4. Materials and Methods ................................................................................................... 27 4.1 Suppliers of chemicals ............................................................................................... 27 4.2 Media and growth conditions for microorganisms .................................................... 27 4.3 Transformation of microorganisms ............................................................................ 27 4.4 Plasmid construction.................................................................................................. 28 4.4.1 General procedure and overview ....................................................................... 28 4.4.2 Full-length cDNA constructs of putative RhoGEFs and RhoGAPs ................... 35 4.4.3 Plasmids encoding MBP- and GST-tagged RhoGEF constructs ...................... 35 4.4.4 Plasmids encoding MBP-tagged GAP constructs ............................................. 36 4.4.5 Plasmids for yeast two-hybrid analyses ............................................................. 37 4.4.6 Plasmids for analysis of subcellular fusion protein localization ......................... 39 4.4.7 Plasmids for overexpression of epitope-tagged fusion proteins for phenotypic ... rescue and coimmunoprecipitation experiments ............................................... 41 4.5 Strains ........................................................................................................................ 42 ii Table of Contents 4.6 General molecular biological techniques .................................................................. 45 4.6.1 Isolation and analysis of nucleic acids ............................................................... 45 4.6.2 Reverse transcription of RNA............................................................................. 45 4.6.3 Polymerase chain reaction (PCR) ...................................................................... 46 4.6.4 DNA agarose gel electrophoresis ...................................................................... 46 4.6.5 Enzymatic restriction and modification of DNA .................................................. 46 4.6.6 Ligation ............................................................................................................... 47 4.7 Biochemical and immunological methods ................................................................. 47 4.7.1 Preparation of N. crassa crude extracts for protein analysis ............................. 47 4.7.2 Separation of proteins by SDS polyacrylamide gel electrophoresis (PAGE) .... 47 4.7.3 Western blotting.................................................................................................. 48 4.7.4 Immunoprecipitation ........................................................................................... 48 4.7.5 Analysis of MAK1 phosphorylation status.......................................................... 49 4.7.6 Protein expression and purification from E. coli ................................................ 50 4.7.7 In vitro GEF activity assays ................................................................................ 51 4.7.8 Copurification experiments ................................................................................. 52 4.8 Yeast two-hybrid assays ............................................................................................ 52 4.9 Microscopy ................................................................................................................. 52 4.10 Bioinformatic tools ..................................................................................................... 53 4.10.1 DNA and protein sequence analysis .................................................................. 53 4.10.2 Phylogenetic analysis and alignment ................................................................. 53 5. Results ............................................................................................................................. 55 5.1 Analysis of Rho regulator specificity in N. crassa ..................................................... 55 5.1.1 Comparison with the yeast Rho regulatory machinery ...................................... 55 5.1.2 Heterologous expression and purification of RhoGAP and RhoGEF ................... constructs ........................................................................................................... 58 5.1.3 In vitro GEF activity assays ................................................................................ 60 5.2 Analysis of the CDC42/RAC/CDC24 module in N. crassa ....................................... 64 5.2.1 Morphological characterization of mutants ........................................................ 64 5.2.2 In vitro GEF activity of mutant CDC24 versions ................................................ 68 5.2.3 Analysis of the subcellular localization of RAC and CDC42 ............................. 71 5.3 Analysis of the RHO1/RHO2/NCU00668 module in N. crassa ................................ 73 5.3.1 Potential autoregulatory effect of the DEP domain on GEF activity of.................. NCU00668 .......................................................................................................... 73 5.3.2 Morphological characterization of rho-1, rho-2 and NCU00668 mutants .......... 75 5.3.3 Effector pathways regulated by RHO1 and RHO2 in N. crassa ........................ 78 5.3.4 Analysis of the
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