CHARACTERIZATION OF NEUROSPORA CELL FUSION EVENTS
FREE, STEPHEN J. Principal Investigator
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Fusion between two eukaryotic cells is a fundamentally interesting and biologically important process.We have only a rudimentary understanding of how cell fusion occurs. This grant proposal focuses oncharacterizing the process of anastomosis (vegetative hyphal cell fusion) in the filamentous fungusNeurospora crassa. The major goals of the proposed research are to identify and characterize proteins thatfunction to mediate the cell fusion event and to determine how they function to facilitate cell fusion. The genes that are required for cell fusion, as defined by cell fusion-defective mutants, will be identifiedand characterized. The mutations responsible for the cell fusion-defective phenotype will be mapped tosmall regions of the Neurospora genetic map by classicalgenetic mapping techniques. The affected geneswill be identified using a PCRamplification and DNAsequencing strategy. The use of this positional cloningapproach has been made possible by the publication of the Neurospora genomic DNA sequence. Theidentity of the genes encoding cell fusion proteins will be verified by gene disruption experiments. Anastomosis can be characterized as a series of steps leading to a cell fusion event. To help usdetermine which proteins function is each of these steps, differential interference contrast microscope andconfocal microscope time-lapse video recording systems will be used to characterize the cell fusion mutantsand elucidate which cell fusion steps are blocked within these mutants. We will also determine the locationand follow the movement of a few key cell fusion proteins during cell fusion events. The cellular location ofcell fusion proteins will be determined in immunolocalization experiments. Keycell fusion proteins will betagged with the green fluorescent protein and the movement of these tagged proteins during cell fusionevents will be followed with the confocal microscope time-lapse video recording system. We anticipate thatthese experiments will provide us with important insights into how cell fusion events are orchestrated.Public Health Relevance: Cell fusion events are critical to the process of fertilization andfor thedifferentiation of muscle, bone, and placenta. The proposed research will help us better understand cellfusion events.