Gamete membrane adhesion and fusion during fertilization

描述（由申请人提供）：我的实验室的长期目标是了解在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特征的细胞生物学事件。这些事件包括：异性配子之间的特异性识别和粘附；粘附诱导的信号转导和配子激活；以及在两个配子的质膜上特异的融合位点之间的粘附和融合。本提案的目的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是FUS1蛋白，它是由mt+编码的，以前被证明是细胞-细胞融合所必需的配子特异性基因。新的序列分析表明FUS1与细菌粘附蛋白具有相似性。我们对内源性FUS1蛋白进行了表征，并确定其约为95 kD，定位于配子激活前未激活的mt+交配结构的一小片膜上。在配子激活后的几秒钟内，FUS1分布在新形成的受精小管的整个表面。此外，我们发现fus1-配子不仅不能进行配子融合，而且它们也不能进行新发现的交配结构粘附步骤，即配子对接。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分析，结合衣藻遗传、生化和分子遗传操作的易用性，现在可以对衣藻的配子质膜粘附/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；鉴定粘附和融合所需的mt+配子特异性蛋白；检测FUS1的功能域；并鉴定和表征mt配子中的粘附和融合蛋白。目前，配子融合的分子机制尚不清楚。了解衣藻的配子融合对未来人类生殖的研究有重要意义。

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.