Function of the protein EFF-1 in cell membrane fusion

描述(由申请人提供)：这项建议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，所有真核生物门在进化过程中都是相同的。然而，细胞融合的分子机制在所有物种中基本上都是未知的。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。我们对线虫线虫的研究发现了Eff-1，这是已知的第一种完全需要和完全特异的细胞膜融合过程的细胞膜蛋白。EFF-1是一种新的I型膜蛋白，其序列基序通过膜脂类物质的直接相互作用和/或处理来暗示作用模式：磷脂酶A2活性部位和潜在的类病毒两亲性融合肽。突变细胞中缺乏功能性的EFF-1，在这一过程的最早阶段就阻止了细胞膜融合。 我们建议通过以下几条途径来确定EFF-1在膜融合中的分子功能，并进一步剖析细胞融合的完整机制：1.通过定向突变分析和酶活性测定来定位和检测EFF-1的功能结构域。2.通过对活胚胎融合细胞内标记的EFF-1蛋白进行成像，评估EFF-1在细胞融合事件的动态结构背景中的作用。3.通过分离与EFF-1在物理和基因上相互作用的分子来鉴定细胞融合机制的其他蛋白质成分。本提案是根据NICHD的PAS-00-067：“膜特性：探索精子/微生物的敏感性”提交的，因为它侧重于PA专门针对的主题。然而，这项拟议研究的科学影响具有普遍重要性。我们的结果将增加对发育细胞融合的分子机制的理解，发育细胞融合是动物繁殖、发育和健康的关键和未被探索的组成部分。

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.