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（申请人提供）：我实验室的长期目标是了解受精过程中配子相互作用和细胞-细胞融合的细胞和分子事件。我们使用单细胞双鞭毛衣原体进行研究。在衣原体受精过程中，相互作用的mt+和mt-配子经历了几乎所有生物体都具有受精特征的细胞生物学事件。这些活动包括：异性配子之间的特异性识别和粘附;粘附诱导的信号转导和配子活化;以及两个配子质膜上的特化融合位点之间的粘附和融合。本提案的目的是描述配子融合的细胞和分子机制。我们的研究重点之一将是FUS 1蛋白，它是由一个mt+，配子特异性基因编码的，以前被证明是细胞-细胞融合所必需的。新的序列分析表明FUS 1与细菌粘附蛋白具有相似性。我们已经表征了内源性FUS 1蛋白，并确定它约为95 kD，并且在配子激活之前定位于未激活的mt+交配结构处的一小片膜。在配子激活后的几秒钟内，FUS 1开始分布在新形成的受精小管的整个表面。此外，我们发现，不仅是fus 1-配子不能进行配子融合，但他们也没有经历一个新确定的，交配结构粘附步骤，称为配子对接。因此，与参与病毒融合的蛋白质非常相似，FUS 1对于质膜结合和质膜融合都是必需的。我们的新试剂和生物测定，结合衣原体的遗传，生物化学和分子遗传操作的便利性，现在可以进行衣原体配子质膜粘附/融合的详细分析。我们的具体目标是表征MT+配子中粘附和融合蛋白的细胞和分子特性;识别粘附和融合所需的MT+配子特异性蛋白;检查FUS 1的功能结构域;以及识别和表征MT-配子中的粘附和融合蛋白。目前，配子融合的分子机制在任何生物中都不清楚。了解配子融合衣原体应告知未来的研究对人类生殖。