Gamete Membrane Adhesion and Fusion During Fertilization

描述(由申请人提供)：在受精过程中，雄配子和雌配子融合形成受精卵是真核生物生命中的决定性时刻。虽然了解配子融合对生殖健康至关重要，但我们甚至还不知道对于单个生物体来说，配子膜融合所需的分子或分子步骤。我的实验室使用双鞭毛、单细胞绿藻莱茵衣藻作为模型系统来研究受精。在受精的第一阶段，正配子和负配子鞭毛之间的粘连将它们聚集在一起，并激活两者以暴露专门用于融合的细胞膜位置。接下来，融合质膜紧密接触并立即融合。在任何生物体中，我们现在第一次证明融合膜的附着和两膜的合并在基因上是可区分的，并且至少由两种不同的整膜蛋白执行。配子膜之间的融合前附着由物种特异性加配子特异性蛋白FUS1控制，随后的膜合并取决于广泛保守的负配子特异性蛋白HAP2。HAP2家族成员存在于海绵、线虫、几种昆虫、高等植物和许多毁灭性的致病原生生物中，包括疟原虫。此外，我们还揭示了膜阻断对多精母细胞的分子机制，表明在衣藻细胞膜阻断到多配子过程中，FUS1和HAP2都经历了快速的、融合依赖的蛋白质分解。这个衣藻系统使我们能够利用其他系统尚未获得的知识和方法来剖析配子融合的分子机制。我们有完善的生物检测方法来检测和量化配子相互作用中的每一步；我们有不育突变在受精的几个步骤中被阻止；有机体很容易受到遗传和分子生物学操作的影响；我们可以制备足够数量的蛋白质，用于生物化学和结构研究。我们发现的HAP2已经在疟疾受精研究中产生了意想不到的影响。与我们的合作者一起，我们证明了HAP2对于疟原虫配子融合和蚊子传播疟疾是必不可少的，因此是疟疾传播阻断疫苗的新的主要靶点。对于至少一个生物体来说，了解配子膜融合反应过程中发生的分子事件将对生殖生物学领域产生重大影响。这些知识将为剖析其他生物体中配子融合的基本原理、开发避孕药具和治疗不孕症建立一个框架。本文提出的研究策略的目的是测试HAP2在膜融合反应中作为融合蛋白的模型。我们将识别与HAP2相互作用的蛋白质，我们将研究HAP2的功能结构域，我们将识别在膜融合过程中发挥作用的新蛋白质。

DESCRIPTION (provided by applicant): Fusion of male and female gametes to form a zygote during fertilization is the defining moment in the life of a eukaryote. Although understanding gamete fusion is critical for reproductive health, we do not yet know for even a single organism the neither molecules nor molecular steps required for fusion of gamete membranes. My laboratory uses the biflagellated, unicellular green alga Chlamydomonas reinhardtii as a model system to study fertilization. In the first phase of fertilization, adhesion between the flagella of plus and minus gametes brings them together and also activates both to expose cell membrane sites specialized for fusion. Next, the fusogenic plasma membranes come into intimate contact and immediately fuse. For the first time in any organism, we have now shown that attachment of fusogenic membranes and merger of the two membranes are genetically distinguishable and are carried out by at least two different integral membrane proteins. Pre- fusion attachment between gamete membranes is governed by a species-specific plus gamete-specific protein FUS1, and subsequent membrane merger depends on a broadly conserved, minus gamete-specific protein, HAP2. HAP2 family members are present in sponges; cnidarians; several insects; higher plants; and many devastating pathogenic protists, including Plasmodium. Furthermore, we have also uncovered a molecular mechanism for a membrane block to polyspermy, demonstrating that both FUS1 and HAP2 undergo rapid, fusion-dependent proteolysis during a Chlamydomonas membrane block to polygamy. This Chlamydomonas system is poised to allow us to dissect the molecular mechanisms of gamete fusion using knowledge and approaches not yet available for other systems. We have well-established bioassays to detect and quantify each step in gamete interactions; we have sterile mutants blocked at several steps in fertilization; the organism is easily amenable to genetic and molecular biological manipulations; and, we can prepare quantities of protein sufficient for biochemistry and structural studies. Our discovery of HAP2 already has had an unexpected impact in malaria fertilization research. With our collaborators we showed that HAP2 is essential for Plasmodium gamete fusion and mosquito transmission of malaria, and therefore a new prime target for a malaria transmission-blocking vaccine. Understanding, for at least one organism, the molecular events that occur during the gamete membrane fusion reaction will have a large impact on the field of reproductive biology. Such knowledge will establish a framework for dissecting fundamental principles of gamete fusion in other organisms and for development of contraceptives and for treating infertility. The objective of the research strategy presented here is to test the model that HAP2 functions as a fusion protein during the membrane fusion reaction. We will identify proteins that interact with HAP2, we will study the functional domains of HAP2, and we will identify new proteins that function during membrane fusion.