The long-term goal of this proposal is to understand the mechanisms by which cell membranes become fusion competent in normal animal development. The fusion of somatic cells to form syncytial tissues is an essential part of human development, and cell fusion is an ancient process, shared in evolution by all eukaryotic phyla. Yet the molecular mechanism of cell fusion is essentially unknown in all species. Finding the molecules that drive plasma membrane fusion is therefore a major priority in understanding human health. Our studies of the nematode C elegans have uncovered EFF-1, the first cellular membrane protein in any species that is known to be completely required for and completely specific to the process of cell membrane fusion. EFF-1 is a novel type I membrane protein, with sequence motifs that suggest modes of action via direct interaction and/or processing of membrane lipids: a phospholipase A2 active site, and a potential virus-like amphipathic fusion peptide. The absence of functional EFF-1 in mutant cells blocks cell membrane fusion at the earliest step in the process. ? ? We propose to determine the molecular function of EFF-1 in membrane fusion, and to further dissect the full mechanism of cell fusion via the following lines of inquiry: 1. Map and test the functional domains of EFF-1 through directed mutation analysis and via assays of enzymatic activity. 2. Assess the role of EFF-1 within the dynamic structural context of a cell fusion event by imaging of labeled EFF-1 protein within the fusing cells of live embryos. 3. Identify other protein components of the cell fusion machinery through isolation of molecules that interact physically and genetically with EFF-1. This proposal is submitted in response to PAS-00-067 from NICHD: """"""""Membrane properties: Exploration of sperm/microbe susceptibility"""""""", as it focuses on topics specifically targeted by the PA. The scientific impact of the proposed research is of general import, however. Our results will increase understanding of the molecular mechanism underlying developmental cell fusion, a crucial and under-explored component of animal reproduction, development, and health.
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