Project Grant 2222841

RUI: PROTEIN-PROTEIN INTERACTIONS OF PROTEIN KINASE C DURING POLARIZED GROWTH IN FILAMENTOUS FUNGI -THIS PROJECT SEEKS TO INCREASE OUR UNDERSTANDING OF HOW FUNGI GROW. FUNGI ARE MICROORGANISMS WHICH ARE USED TO PRODUCE COMMERCIALLY AND MEDICINALLY VALUABLE PRODUCTS, WHILE OTHERS CAUSE DISEASE AND RESULT IN MAJOR FINANCIAL LOSSES DUE TO CROP DAMAGE AND SPOILAGE OF STORED FOODS. UNDERSTANDING THEIR GROWTH HELPS TO ADVANCE TECHNOLOGIES INVOLVING THEIR USE AND AIDS IN DEVELOPING STRATEGIES TO CONTROL THEIR SPREAD IN HARMFUL CONTEXTS. FUNGI EXIST IN TWO DIFFERENT FORMS; SPHERICAL, SINGLE-CELLED ORGANISMS AND ELONGATED, MULTICELLULAR ORGANISMS, WHICH ARE CALLED FILAMENTOUS FUNGI. THIS PROJECT FOCUSES ON GROWTH AND CELL DIVISION IN FILAMENTOUS FUNGI. IN ORDER TO GROW AND COLONIZE THEIR HOSTS, FILAMENTOUS FUNGI EXTEND INTO THEIR ENVIRONMENTS BY ADDING CELLULAR MATERIALS AT THE TIPS OF GROWING FILAMENTS, AND THEY DIVIDE BY CONSTRUCTING CROSS-WALLS, CALLED SEPTA, AT EVENLY SPACED INTERVALS ALONG THE FILAMENTS. THIS PROJECT BUILDS ON PREVIOUS NSF FUNDED RESEARCH IN THIS LABORATORY WHICH IDENTIFIED PROTEINS INVOLVED IN FILAMENTOUS FUNGAL GROWTH AND CELL DIVISION, AND BEGAN TO DETERMINE WHICH GROWTH-RELATED PROTEINS FORM PHYSICAL COMPLEXES THAT ARE INVOLVED IN GROWTH AND DIVISION. ONGOING WORK WILL EXPAND GROWTH AND CELL DIVISION PROTEIN COMPLEXES BY IDENTIFYING NEW PROTEINS WHICH ARE INVOLVED, AND IT WILL FURTHER DEFINE HOW THESE COMPLEXES FUNCTION. THIS RESEARCH WILL BE CARRIED OUT BY TWO SENIOR SCIENTISTS AT RHODES COLLEGE (MEMPHIS, TN) WORKING WITH UNDERGRADUATE STUDENTS ENROLLED AT RHODES AND STUDENTS ATTENDING HISTORICALLY BLACK COLLEGES IN THE MEMPHIS REGION. UNDERGRADUATE STUDENTS WILL BE INTEGRALLY INVOLVED IN THE WORK, AND THIS RESEARCH EXPERIENCE WILL STRENGTHEN THEIR SCIENTIFIC EDUCATION. THIS PROJECT INVESTIGATES THE FUNCTION OF PROTEIN KINASE C IN FILAMENTOUS FUNGAL GROWTH AND CELL DIVISION (SEPTATION). USING THE FILAMENTOUS FUNGUS ASPERGILLUS NIDULANS AS A MODEL ORGANISM PROTEINS INVOLVED IN GROWTH AND CELL DIVISION, INCLUDING THE A. NIDULANS HOMOLOG OF PROTEIN KINASE C, PKCA, HAVE BEEN IDENTIFIED. PREVIOUS RESEARCH BY THE PRIMARY INVESTIGATORS OF THIS PROJECT USED TECHNIQUES INCLUDING IN VIVO MICROSCOPY OF FLUORESCENCE LABELED PROTEINS, IMMUNOPRECIPITATIONS FOLLOWED BY MASS SPECTROMETRY AND PROTEOMICS ANALYSES, AND YEAST TWO-HYBRID ASSAYS TO IDENTIFY SEVERAL PROTEINS THAT PHYSICALLY INTERACT WITH PKCA INCLUDING THE FORMIN SEPA, THREE RHO-TYPE GTPASE ORTHOLOGS, A CHITIN SYNTHASE ORTHOLOG, A GLUCAN SYNTHASE, AND TWO IMPORTANT SCAFFOLD PROTEINS - A. NIDULANS IQGAP ORTHOLOG SEPG AND AN A. NIDULANS PAXILLIN ORTHOLOG PAXB - WHICH APPEAR TO HAVE ROLES IN PKCA?S LOCALIZATION TO SEPTATION SITES. THIS PROJECT WILL DETERMINE IF THESE PROTEINS OR A SUBSET ARE PKCA SUBSTRATES, AND IF PHOSPHORYLATION EVENTS PLAY IMPORTANT ROLES IN GROWTH AND CELL DIVISION. THE MECHANISMS BY WHICH PKCA PROTEIN COMPLEXES COALESCE WILL BE EXPLORED BY IDENTIFYING KEY DOMAINS OF PKCA AND COMPLEXED PROTEINS THAT FACILITATE PROTEIN-PROTEIN INTERACTIONS AMONG THEM. NOT ONLY WILL THIS RESEARCH BE OF VALUE TO THE FILAMENTOUS FUNGI COMMUNITY, BUT IT WILL ALSO BENEFIT THE BROADER CELL BIOLOGY COMMUNITY, AS IT WILL SHED LIGHT ON THE FACTORS THAT AFFECT PROTEIN KINASE C RECRUITMENT (AND PROTEIN RECRUITMENT IN GENERAL) TO PROTEIN COMPLEXES AND HOW PROTEIN NETWORKS FUNCTION SUBSEQUENT TO RECRUITMENT. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.