Gamete membrane adhesion and fusion during fertilization

受精过程中配子膜的粘附和融合

• 批准号: 6752066
• 负责人: William J Snell
• 金额: $ 25.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752066
• 关键词: Chlamydomonas; SDS polyacrylamide gel electrophoresis; actins; cell adhesion; cell fusion; cell membrane; fertilization; fungal proteins; germ cells; immunoprecipitation; protein binding; protein localization; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): The long-term goals of my laboratory are to understand the cellular and molecular events that underlie gamete interactions and cell-cell fusion during fertilization. We use the unicellular, biflagellated alga Chlamydomonas for our studies. During Chlamydomonas fertilization, the interacting mt+ and mt- gametes undergo the cell biological events that characterize fertilization in almost all organisms. These events include: specific recognition and adhesion between gametes of opposite sex; adhesion-induced signal transduction and gamete activation; and adhesion and fusion between specialized fusogenic sites on the plasma membranes of the two gametes. The objectives of this proposal are to characterize the cellular and molecular mechanisms of gamete fusion. One focus of our studies will be on the FUS1 protein, which is encoded by an mt+, gamete-specific gene previously shown to be essential for cell-cell fusion. New sequence analysis indicates that FUS1 exhibits similarity to bacterial adhesion proteins. We have characterized the endogenous FUS1 protein and determined that it is approximately 95 kD and is localized to a small patch of membrane at the unactivated mt+ mating structure before gamete activation. Within seconds after gamete activation, FUS1 becomes distributed over the entire surface of the newly formed fertilization tubule. In addition, we discovered that not only are fus1- gametes unable to undergo gamete fusion, but they also fail to undergo a newly identified, mating structure adhesion step, called gamete docking. Thus, much like proteins involved in viral fusion, FUS1 is essential both for plasma membrane binding and plasma membrane fusion. Our new reagents and bioassays, in conjunction with the ease of genetic, biochemical, and molecular genetic manipulations in Chlamydomonas, now make it possible to carry out a detailed analysis of gamete plasma membrane adhesion/fusion in Chlamydomonas. Our specific aims are to characterize the cellular and molecular properties of adhesion and fusion proteins in mt+ gametes; to identify mt+ gamete-specific proteins required for adhesion and fusion; to examine the functional domains of FUS1; and to identify and characterize adhesion and fusion proteins in mt- gametes. Currently, the molecular mechanisms of gamete fusion are not understood in any organism. Understanding gamete fusion in Chlamydomonas should inform future studies on human reproduction.

描述（由申请人提供）：我实验室的长期目标是了解施肥过程中配子相互作用和细胞融合的细胞和分子事件。我们将单细胞，双叶藻藻藻衣原体用于我们的研究。在衣原体受精过程中，相互作用的MT+和MT配子会经历几乎所有生物体受精的细胞生物事件。这些事件包括：异性配子之间的具体认识和粘附；粘附引起的信号转导和配子激活；两个配子的质膜上的专门融合位点之间的粘附和融合。该提案的目标是表征配子融合的细胞和分子机制。我们研究的一个重点将放在FUS1蛋白上，该蛋白是由MT+的配子特异性基因编码的，先前证明是细胞细胞融合所必需的。新的序列分析表明，FUS1表现出与细菌粘附蛋白相似。我们已经表征了内源性FUS1蛋白，并确定它大约是95 kD，并在配子激活之前的未激活MT+交配结构处定位于一小块膜。在配子激活后的几秒钟内，FUS1分布在新形成的受精管的整个表面上。此外，我们发现不仅FUS1-配子无法进行配子融合，而且它们也未能经历新鉴定的交配结构粘附步骤，称为配子对接。因此，就像参与病毒融合的蛋白质一样，FUS1对于质膜结合和质膜融合都是必不可少的。我们的新试剂和生物测定法，结合了衣原体中的遗传，生化和分子遗传操作的易感性，现在可以对衣原体中的配子质膜粘附/融合进行详细分析。我们的具体目的是表征MT+配子中粘附和融合蛋白的细胞和分子特性。确定粘附和融合所需的MT+配子特异性蛋白；检查FUS1的功能域；并识别和表征MT-配子中的粘附和融合蛋白。目前，在任何生物体中都不了解配子融合的分子机制。了解衣原体中的配子融合应为未来的人类生殖研究提供信息。