Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTScan exert sufficient force to penetrate human epithelial tissue without the need for secreted enzyme activity. This is consistent with the observed hyphalpenetration of medical-grade silicone, which has a similar Young’s modulus to human cartilage.112. Cdc14 association with basal bodies in the oomycete Phytophthora infestans indicates potential new role for this protein phosphatase. AudreyM.V. Ah-Fong, Howard S. Judelson. Plant Pathology & Microbiology, University of California, Riverside, CA.The dual-specificity phosphatase Cdc14 is best known as a regulator of cell cycle events such as mitosis and cytokinesis in yeast and animal cells.However, the diversity of processes affected by Cdc14 in different eukaryotes raises the question of whether its cell cycle functions are truly conservedbetween species. Analyzing Cdc14 in Phytophthora infestans should provide further insight into the role of Cdc14 since this organism does not exhibit aclassical cell cycle. Prior study in this organism already revealed novel features of its Cdc14. For example, instead of being post-translationally regulatedlike its fungal and metazoan relatives, PiCdc14 appears to be mainly under transcriptional control. It is absent in vegetative hyphae where mitosis occursand expressed only during the spore stages of the life cycle which are mitotically quiescent, in contrast to other systems where it is expressedconstitutively. Since transformants overexpressing PiCdc14 exhibit normal nuclear behavior, the protein likely does not play a critical role in mitoticprogression although PiCdc14 is known to complement a yeast Cdc14 mutation that normally arrests mitosis. Further investigation into the role of PiCdc14uncovered a novel role. Subcellular localization studies based on fusions with fluorescent tags showed that PiCdc14 first appeared in nuclei during earlysporulation. During the development of biflagellated zoospores from sporangia, PiCdc14 transits to basal bodies, which are the sites from which flagelladevelop. A connection between Cdc14 and flagella is also supported by their phylogenetic distribution, suggesting an ancestral role of Cdc14 in basalbodies and/or flagellated cells. To help unravel the link between PiCdc14 and the flagella apparatus, searches for its interacting partners using both yeasttwo hybrid and affinity purification are underway. Together with colocalization studies involving known basal body/centrosome markers such as centrinand gamma-tubulin, the location and hence the likely roles of PiCdc14 will be revealed.113. Colletotrichum orbiculare Bub2-Bfa1 complex, a spindle position checkpoint (SPOC) component in Saccharomyces cerevisiae, is involved in properprogression of cell cycle. Fumi Fukada 1 , Ayumu Sakaguchi 2 , Yasuyuki Kubo 1 . 1) Laboratory of Plant Pathology, Graduate School of Life and EnvironmentalSciences, Kyoto Prefectural University, Kyoto, Japan; 2) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.Colletotrichum orbiculare is an ascomycete fungus that causes anthracnose of cucumber. In Saccharomyces cerevisiae, the orientation of the mitoticspindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. A surveillance mechanism named spindle position checkpoint(SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-daughter polarity axis. BUB2 is a component of SPOC andconstitutes the main switch for the mitotic exit network (MEN) signaling. We identified and named this homolog as CoBUB2 in C. orbiculare and generatedgene knock-out mutants. First, we observed morphogenesis and pathogenesis of the cobub2 mutants. The cobub2 mutants formed abnormal appressoriaand penetration hyphae on model substrates, and the cobub2 mutants also showed attenuate pathogenesis to cucumber leaves. Second, we observedmitosis based on mitotic spindle behavior and nuclear DAPI staining during appressorium development. In the wild type, mitosis occurred in appressoriumdeveloping conidia after 4h incubation, whereas interestingly, in the cobub2 mutants, mitosis occurred in pre-germinated conidia after 2h incubation.After development of appressorium, in some germlings the daughter nucleus was delivered from conidia to appressoria, and the others perform secondround of mitosis in appressorium developing conidia after 4h incubation. Third, we evaluated the timing of S phase and M phase during appressoriumdevelopment in wild type and the cobub2 mutants by cell cycle specific inhibitors. In the cobub2 mutants, it was shown that the transition period from G1phase to S phase accelerated about 2h than that of the wild type. Last, in S. cerevisiae, Bub2 forms GTPase activating protein (GAP) complex with Bfa1, andBub2-Bfa1 GAP complex constitutes SPOC. Then we named homolog of BFA1 as CoBFA1 in C. orbiculare and generated cobfa1 mutants. From observationof nuclear division, the cobfa1 mutants showed similar behavior of nuclear division to the cobub2 mutants. Therefore, it is assumed that CoBub2 formsGAP complex with CoBfa1, however, CoBub2-CoBfa1 GAP complex has different function from that in S. cerevisiae maintaining G1 phase duration orsetting up the proper time of S phase.114. Metazoan-like mitotic events in the basidiomycetous budding yeast Cryptococcus neoformans - a human fungal pathogen. L. Kozubowski 1,2 , V.Yadav 3 , G. Chatterjee 3 , M. Yamaguchi 5 , I. Bose 4 , J. Heitman 2 , K. Sanyal 3 . 1) Department of Medicine, Division of Infectious Diseases, Duke UniversityMedical Center, Durham, NC, USA; 2) Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA; 3)Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India; 4)Department of Biology, Western Carolina University, Cullowhee, NC, USA; 5) Medical Mycology Research Center, Chiba University, Chiba, Japan.Mitosis in ascomycetous budding yeasts is characterized by several features that are distinct from those of metazoans. In Saccharomyces cerevisiae,centromeres are always clustered in a single spot, the kinetochores are fully assembled for the majority of the cell cycle, and the nuclear envelope (NE)does not break down (closed mitosis). Currently it is not clear how these mechanisms evolved or whether these features are a universal characteristichallmark of the budding mode of cellular division. Here we report an analysis of key mitotic events in the basidiomycetous human fungal pathogenCryptococcus neoformans. The dynamics of microtubules, the kinetochore, NE and the nucleolus were analyzed by time-lapse microscopy usingfluorescently tagged proteins. In striking contrast to ascomycetous budding yeast, centromeres in C. neoformans were not clustered in non-dividing cells.Prior to mitosis, centromeres underwent gradual clustering, eventually forming a single spot, which then migrated into the daughter cell where thechromosomal division occurred. One set of chromosomes migrated back to the mother cell and subsequent de-clustering of centromeres occurred in bothcells. Analysis of individual components of the kinetochore indicated that kinetochores assemble in a step-wise manner in C. neoformans. While the innerkinetochore (Cse4, Mif2) was present throughout the entire cell cycle, the middle kinetochore (Mtw1) assembled prior to mitosis when centromeresunderwent clustering, and this was then followed by assembly of the outer kinetochore (Dad1, Dad2). Formation of the outer kinetochore during mitosis,as observed in metaozoans that undergo an open mitosis, prompted us to examine the fate of the NE at various cell cycle stages. Several lines of evidencesuggested that C. neoformans undergoes a semi-open mitosis. The nuclear pore marker GFP-Nup107, and a nucleolar marker GFP-Nop1 dispersed into thecytoplasm during metaphase, a nuclear membrane marker Ndc1 exhibited a localization pattern that also suggests a partial opening of the NE duringmitosis. A semi-open mitosis was further confirmed by transmission electron microscopy. In summary, our data demonstrate that key mitotic events in C.neoformans are similar to that of metazoan cells. This study sheds new light on the evolution of mitosis during fungal speciation.115. Distinctive Mitotic Localization of a Novel Suppressor of nimA1 Provides New Insight into NIMA Function. Jennifer R. Larson, Stephen A. Osmani.Department of Molecular Genetics, The Ohio State University, Columbus, OH.The NIMA kinase is an essential regulator of mitotic events in Aspergillus nidulans. Not only is NIMA essential for initiating mitosis its overexpression canprematurely induce mitotic events including DNA condensation and nuclear pore complex (NPC) disassembly in A. nidulans and human cells. One of thekey roles for NIMA at the onset of mitosis is its regulation of NPCs. A previous study aimed at identifying suppressors of the temperature-sensitive nimA127th Fungal Genetics Conference | 149