Function of the protein EFF-1 in cell membrane fusion

描述（由申请人提供）：本提案的长期目标是了解细胞膜在正常动物发育中成为融合能力的机制。体细胞融合形成合胞体组织是人类发育的重要组成部分，细胞融合是一个古老的过程，在所有真核生物门的进化中共享。然而，在所有物种中，细胞融合的分子机制基本上是未知的。因此，寻找驱动质膜融合的分子是了解人类健康的主要优先事项。我们在线虫C elegans的研究中发现了ef -1，这是已知的所有物种中第一个完全需要并且完全特定于细胞膜融合过程的细胞膜蛋白。ef -1是一种新型的I型膜蛋白，其序列基序提示其通过直接相互作用和/或膜脂加工的作用模式：磷脂酶A2活性位点和潜在的病毒样两亲性融合肽。突变细胞中功能性ef -1的缺失阻碍了细胞膜融合过程的早期阶段。

DESCRIPTION (provided by applicant): 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.