Function of the protein EFF-1 in cell membrane fusion

这项提议的长期目标是了解细胞膜 在正常的动物发育中具有融合能力。体细胞的融合形成 合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， 所有真核生物门在进化上都是相同的。然而，细胞融合的分子机制基本上是 在所有物种中都是未知的。因此，寻找驱动质膜融合的分子是一个 了解人类健康的主要优先事项。我们对线虫的研究有 发现了Eff-1，这是已知的任何物种中第一个完全 这是细胞膜融合过程所必需的，也是完全特异的。EFF-1是一种新型的I型 膜蛋白，具有通过直接相互作用和/或暗示作用模式的序列基序 膜脂的加工：磷脂酶A2活性部位和潜在的病毒样 两亲性融合肽。突变细胞中功能缺失的Eff-1阻断细胞膜 在这一过程中最早的一步是融合。 我们建议确定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通过以下几个方面的探讨：1.映射和测试功能 通过定向突变分析和酶活性测定确定EFF-1的结构域。2.评估 通过标记成像研究EFF-1在细胞融合事件动态结构中的作用 活胚胎融合细胞内的Eff-1蛋白。3.确定细胞的其他蛋白质成分 通过分离与EFF-1在物理和基因上相互作用的分子来实现核聚变机制。 本提案是根据NICHD的PAS-00-067提出的：“膜特性： 探索精子/微生物敏感性“，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背后的分子机制的理解，这是一个至关重要的 以及动物繁殖、发育和健康的未被充分开发的组成部分。

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.