Gamete membrane adhesion and fusion during fertilization

描述(申请人提供)：我的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-细胞融合背后的细胞和分子事件。我们使用单细胞、双鞭毛藻衣藻进行研究。在衣藻受精过程中，相互作用的mt+和mt配子经历了几乎所有生物受精的细胞生物学事件。这些事件包括：异性配子之间的特异性识别和黏附；黏附诱导的信号转导和配子激活；以及两个配子质膜上特定融合位点之间的黏附和融合。这项建议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FUS1蛋白，它是由先前被证明对细胞-细胞融合至关重要的mt+配子特异性基因编码的。新的序列分析表明，FUS1与细菌黏附蛋白具有相似性。我们已经鉴定了内源FUS1蛋白，并确定它约为95kD，在配子激活之前定位于未激活的mt+交配结构的一小块膜上。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FUS1-配子不仅不能进行配子融合，而且也不能经历新发现的交配结构粘连步骤，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，结合衣藻的遗传、生化和分子遗传操作的简便性，现在使得对衣藻配子质膜黏附/融合的详细分析成为可能。我们的具体目标是确定mt+配子中黏附和融合蛋白的细胞和分子特性；鉴定mt+配子中黏附和融合所需的特异蛋白质；研究FUS1的功能结构域；以及鉴定和表征mt配子中的黏附和融合蛋白。目前，配子融合的分子机制在任何生物体中都不清楚。了解衣藻中的配子融合将有助于未来对人类生殖的研究。

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.