Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSAspergillus nidulans septin interactions and post-translational modifications. Yainitza Hernandez-Rodriguez 1 , Shunsuke Masuo 2 , Darryl Johnson 3 , RonOrlando 3,4 , Michelle Momany 1 . 1) Plant Biology, University of Georgia, Athens, GA, US; 2) Laboratory of Advanced Research A515, Graduate School of Lifeand Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, JP; 3) Department of Chemistry, University of Georgia, Athens, GA, US; 4)Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, US.Septins are cytoskeletal elements found in fungi, animals, and some algae, but absent in higher plants. These evolutionarily conserved GTP bindingproteins form heteroligomeric complexes that seem to be key for the diverse cellular functions and processes that septins carry out. Here we usedAspergillus nidulans, a model filamentous fungus with well defined vegetative growth stages to investigate septin-septin interactions. A. nidulans has fiveseptins: AspA/Cdc11, AspB/Cdc3, AspC/Cdc12 and AspD/Cdc10 are orthologs of the “core-filament forming-septins” in S. cerevisiae; while AspE is onlyfound in filamentous fungi. Using S-tag affinity purification assays and mass spectrometry we found that AspA, AspB, AspC and AspD strongly interact inearly unicellular and multicellular vegetative growth. In contrast, AspE appeared to have little or no interactions with core septins in unicellular stagesbefore septation. However, after septation AspE interacted with other septins, for which we postulate an accessory role. AspE localized to the cortex ofactively growing areas and to septa, and localizations are dependent on other septin partners. Interestingly, core septin localizations can also depend onaccessory septin AspE, particularly post-septation. In addition, LC-MS/MS showed acetylation of lysine residues in AspA before septation and AspC afterseptation. Western blot analysis using an anti-acetylated lysine antibody showed that AspC is highly acetylated in all stages examined, while other septinsshowed acetylation post-septation. Though LC-MS analysis failed to detect phosphorylation of septins, this modification has been widely reported in fungalseptins. Using phosphatase treatments and Western Bloting, we found phosphorylation of AspD, but no other septins. This is interesting because AspDbelongs to a special group of septins that lack a C-terminal coiled-coil found in other septins. However, septin localization is not affected by the absence ofAspD/Cdc10, but by the absence of filamentous fungi specific septin AspE. Our data suggests that septin interactions and modifications change duringdevelopment and growth in A. nidulans, and that some modifications are septin specific.Altered Ras1 trafficking impairs the pathogencity of Cryptococcus neoformans. Connie B. Nichols, Teresa O'Meara, Kyla Selvig, Sandra Breeding, J.Andrew Alspaugh. Dept. of Medicine, Duke University Medical Center, Durham, NC, USA.Cryptococcus neoformans is an opportunistic human fungal pathogen. The ability to cause disease is linked to several different determinants, one ofwhich is the ability to grow at high temperature. Previously we found that one branch of the Ras1 signaling cascade mediates cell morphology andcytokinesis in response to mild stress, such as growth at high temperature. Inactivation of Ras1 and other components of this signaling branch negativelyimpacts C. neoformans pathogenicity. Additionally, this branch of the Ras1 signaling cascade is dependent on the trafficking of Ras1 from theendomembranes to the plasma membrane and is mediated by palmitoylation of the Ras1 protein. We have identified and characterized several C.neoformans protein acyltransferases (PATs), the enzymes responsible for palmitoylation, to further understand the role of palmitoylation and traffickingon Ras1 function and activity during high temperature growth and pathogenesis. Although there is some degree of functional redundancy in this proteinfamily, we identified individual PATs that are required for stress response and virulence in models of cryptococcal disease.Quantification of the thigmotropic response of Neurospora crassa to microfabricated slides with ridges of defined height and topography. KarenStephenson 1 , Fordyce Davidson 2 , Neil Gow 3 , Geoffrey Gadd 1 . 1) Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee,United Kingdom; 2) Division of Mathematics, University of Dundee, Dundee, United Kingdom; 3) Institute of Medical Sciences, University of Aberdeen,Aberdee, United Kingdom.Thigmotropism is the ability of an organism to exhibit an orientation response to a mechanical stimulus. We have quantified the thigmotropic responseof Neurospora crassa to microfabricated slides with ridges of defined height and topography. We show that mutants that lack the formin BNI-1 and theRho-GTPase CDC-42, an activator of BNI-1, had an attenuated thigmotropic response. In contrast, null mutants that lacked cell end-marker protein TEA-1and KIP-A, the kinesin responsible for its localisation, exhibited significantly increased thigmotropism. These results indicate that vesicle delivery to thehyphal tip via the actin cytoskeleton is critical for thigmotropism. Disruption of actin in the region of the hyphal tip which contacts obstacles such as ridgeson microfabricated slides may lead to a bias in vesicle delivery to one area of the tip and therefore a change in hyphal growth orientation. This mechanismmay differ to that reported in Candida albicans in so far as it does not seem to be dependent on the mechanosensitive calcium channel protein Mid1. TheN. crassa Dmid-1 mutant was not affected in its thigmotropic response. Although it was found that depletion of exogenous calcium did not affect thethigmotropic response, deletion of the spray gene, which encodes an intracellular calcium channel with a role in maintenance of the tip-high calciumgradient, resulted in a decrease in the thigmotropic response of N. crassa. This predicts a role for calcium in the thigmotropic response. Our findingssuggest that thigmotropism in C. albicans and N. crassa are similar in being dependent on the regulation of the vectorial supply of secretory vesicles, butdifferent in the extent to which this process is dependent on local calcium-ion gradients.Dynein drives oscillatory nuclear movements in the phytopathogenic fungus Ashbya gossypii and prevents nuclear clustering. S. Grava, M. Keller, S.Voegeli, S. Seger, C. Lang, P. Philippsen. Biozentrum, Molecular Microbiology, University of Basel, CH 4056 Basel, Switzerland.In the yeast Saccharomyces cerevisiae the dynein pathway has a specific cellular function. It acts together with the Kar9 pathway to position the nucleusat the bud neck and to direct the pulling of one daughter nucleus into the bud. Nuclei in the closely related multinucleated filamentous fungus Ashbyagossypii are in continuous motion and nuclear positioning or spindle orientation is not an issue. A. gossypii expresses homologues of all components of theKar9/Dyn1 pathway, which apparently have adapted novel functions. Previous studies with A. gossypii revealed autonomous nuclear divisions and,emanating from each MTOC, an autonomous cytoplasmic microtubule (cMT) cytoskeleton responsible for pulling of nuclei in both directions of the hyphalgrowth axis. We now show that dynein is the sole motor for bidirectional movements. Surprisingly, deletion of Kar9 shows no phenotype. Dyn1, thedynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are involved in the dynamic distributionof nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. Like inbudding yeast, we found that dynein is delivered to cMT +ends, and its activity or processivity is probably controlled by dynactin and Num1. Together withits role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combineddynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.52