Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSFriday, March 15 3:00 PM–6:00 PMChapelCell Wall, Polarity and Hyphal Tip GrowthCo-chairs: Stephan Seiler and Ernestina Castro-LongoriaThe function of Rho type small GTPases for cell polarity in Ustilago maydis. Britta Tillmann 1 , Michaela Wehr 1 , Sonja Frieser 1 , Kay Oliver Schink 2 , JohannesFreitag 1 , Michael Bölker 1 . 1) Dept Biol, Univ Marburg, Marburg, Germany; 2) Institute for Cancer Research, the Norwegian Radium Hospital, Oslo UniversityHospital, Montebello, 0310 Oslo, Norway.Establishment of cell polarization requires the coordinated transport and localized fusion of secretory vesicles. This process is controlled by Rho-typeGTPases that act as molecular switches. Temporal and spatial activation of Rho-GTPases depends on specific guanine nucleotide exchange factors (GEFs).Inactivation of Rho proteins is achieved via interaction with GTPase activating proteins (GAPs) that stimulate the low intrinsic GTPase activity of Rhoproteins. During its life cycle, U. maydis switches between budding and filamentous growth. The Rho type GTPase Rac1 is the main regulator of thismorphogenic transition. The highly related Cdc42 is required for cell separation after mitosis and for formation of retraction septa during filamentousgrowth. We could show that the activator of Rac1, the Rho-GEF Cdc24 is subject to a negative autoregulatory feedback loop. Active Rac1 triggers Cla4dependent multisite phosphorylation of a C-terminal destruction box. This results in rapid degradation of Cdc24 and release of a ternary complexcontaining active Rac1, the scaffold protein Bem1 and the Rac1 effector kinase Cla4. The active Rac1 is subsequently inactivated by GAPs that localize in aring-like fashion underneath of the tip. Both destruction of Cdc24 and inactivation of Rac1 serve to delimit Rac1 activity to the very tip of the fungal hypha.Sustained polarized growth is further supported by recycling of inactive Rac1 to the hyphal tip. This is achieved either by interaction with the Rho proteinGDP dissociation inhibitor Gdi1 or via endocytosis. Active Rho-GTPases recruit specific effectors that trigger the localized fusion of secretory vesicles at thehyphal tip. We found that both Cdc42 and Rac1 interact with Sec3, a subunit of the multiprotein exocyst complex. We have identified several homologs ofexocyst subunits in U. maydis and tested them for functions during polar growth. We could demonstrate that Rac1 is critical for proper localization of theexocyst landmark protein Sec3. We have identified the U. maydis homolog of Smg-GDS, an unconventional activator of Rho GTPases in mammals. Smg-GDS contains a number of armadillo repeats and interacts with both Cdc42 and Rac1. Deletion of the Smg-GDS gene reduces significantly mating andfilament formation, indicating that it contributes to regulation of cell polarity.A quantitative model of hyphal tip growth based on the spatial distribution of exocyst subunits in the human fungal pathogen Candida albicans. DavidCaballero- Lima, Ilyana Kaneva, Simon Watton, C. Jeremy Craven, Peter Sudbery. Dept Molecular Biol & Biotech, Sheffield Univ, Sheffield, S Yorkshire,United Kingdom.We present a quantitative three dimensional treatment of fungal hyphal growth which adapts previous theoretical treatments in the light of advances inour knowledge of the components of polarised growth and their location as revealed by GFP fusions. The model is based on the proposition that vesiclesfuse with the hyphal tip at a rate determined by the experimentally observable local density of exocyst components. Enzymes such as b-1,3 glucansynthase are embedded in the plasma membrane by this process and continue to synthesize cell wall until they are removed from the membrane byendocytosis. The time development of the spatial distribution of the synthase molecules arises from the model. We test the model in the hyphae of thehuman fungal pathogen Candida albicans by quantitative measurements of the distribution of exocyst components and membrane components such asGFP-Rho1, the regulatory subunit of b1,3 glucan synthase, Rom2-GFP, the GEF for Rho1, and the location of actin cortical patches. We show that thepredicted shape and width of the hyphae are in good agreement with that predicted by the model, provided that endocytosis acts to remove cell wallsynthesizing enzymes at the subapical band of cortical actin patches. Thus the pattern of tip growth of fungal hyphae can be satisfactorily explained by asimple but quantitative model rooted within the known molecular processes of polarized growth. At the same time the model exposes the areas ofuncertainty which need to be addressed by future experimentation.Cell wall integrity signaling in Aspergillus fumigatus. Johannes Wagener, Karl Dichtl, Christoph Helmschrott, Sweta Samantaray, Franziska Dirr, MichaelNeubauer. Max von Pettenkofer-Institut, University of Munich, Munich, Germany.Aspergillus fumigatus is an opportunistic pathogen and the most frequent cause of a severe invasive infection termed invasive aspergillosis. Similar toother fungi, this mold is surrounded by a robust cell wall that defines its shape and protects it from physical stress. We have characterized the cell wallintegrity (CWI) pathway of A. fumigates. It comprises at least three major membrane anchored cell wall stress sensors with partially overlapping functions(Wsc1, Wsc3 and MidA), the guanine nucleotide exchange factor Rom2, a Rho GTPase, protein kinase C and a MAP kinase signaling module. We haveshown that the principal CWI components are well conserved from yeasts to filamentous fungi. Though, the importance of the individual components forthe fungal physiology, e.g., cell polarity and conidiation, may significantly differ. Our data stress the importance of the CWI pathway for the antifungal drugsusceptibility and virulence of this pathogen.Optimization of polarity establishment through coupling of multiple feedback loops. Roland Wedlich-Soldner 1 , Tina Fresisinger 1 , Ben Kluender 2 , NikolaMueller 1 , Gisela Beck 1 , Garwin Pichler 4 , Jared Johnson 3 , Richard Cerione 3 , Erwin Frey 2 . 1) Cellular Dynamics adn Cell Patterning, Max Planck Institute ofBiochemistry, Martinsried, Germany; 2) Arnold Sommerfeld Center for Theoretical Physics, Ludwig Maximilians University Munich, Munich, Germany; 3)Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY, USA; 4) Department of Biology II, Ludwig MaximiliansUniversity Munich, Martinsried, Germany.Establishment of cell polarity - or symmetry breaking - relies on local accumulation of polarity regulators. While simple positive feedback is sufficient todrive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. By integrating mathematical modeling with quantitativeexperimental validations we now show that in the yeast Saccharomyces cerevisiae only a combination of actin- and Guanine nucleotide DissociationInhibitor (GDI)-dependent recycling of the central polarity regulator Cdc42 is capable of establishing robust cell polarity at a single site during yeastbudding. The GDI pathway consistently generates a single polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and istherefore highly sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but27th Fungal Genetics Conference | 67