Gamete Membrane Adhesion and Fusion During Fertilization

项目摘要 在受精过程中，雄性和雌性配子融合形成受精卵，这是一个生命的决定性时刻。 真核生物虽然了解配子融合对生殖健康至关重要，但我们还不知道 即使是单个生物体，配子膜融合所需的分子或分子步骤也是如此。我 一个实验室使用双鞭毛单细胞绿色衣藻作为模型系统来研究 受精在受精的第一阶段，正配子和负配子的鞭毛之间的粘附， 它们结合在一起，并激活两者以暴露专门用于融合的细胞膜位点。接下来 融合质膜紧密接触并立即融合。有史以来第一次 我们现在已经表明，融合膜的附着和两种膜的合并 在遗传上是可区分的，并且由至少两种不同的整合膜蛋白进行。预处理 配子膜之间的融合附着是由一种物种特异性蛋白和配子特异性蛋白控制的 FUS 1和随后的膜合并依赖于一种广泛保守的负配子特异性蛋白， HAP2。HAP 2家族成员存在于海绵、刺胞动物、几种昆虫、大多数高等植物中;以及 包括疟原虫在内的许多毁灭性病原原生生物。此外，我们还发现了一个 膜阻断多精受精的分子机制，表明FUS 1和HAP 2都经历了 快速，融合依赖性蛋白水解在衣原体膜阻滞一夫多妻制。这 衣原体系统准备让我们解剖配子融合的分子机制， 其他系统尚不具备的知识和方法。我们有完善的生物检测方法 并量化配子相互作用的每一步;我们有不育突变体在受精的几个步骤中被阻断; 该生物体易于进行遗传和分子生物学操作;并且，我们可以制备 足够用于生物化学和结构研究的蛋白质量。我们对HAP 2的发现 对疟疾受精研究产生了意想不到的影响。与我们的合作者一起，我们证明了HAP 2是 疟原虫配子融合和蚊子传播疟疾必不可少，因此是一种新的主要 疟疾传播阻断疫苗的目标。至少对一种生物来说， 在配子膜融合反应期间发生的事件将对免疫学领域产生重大影响。 生殖生物学这些知识将建立一个框架，剖析基本原则， 配子在其他生物体中的融合以及用于避孕药的开发和用于治疗不育症。的 本文提出的研究策略的目的是测试HAP 2作为融合蛋白发挥作用的模型 在膜融合反应期间。我们将鉴定与HAP 2相互作用的蛋白质，我们将研究 HAP 2的功能域，我们将确定新的蛋白质，在膜融合过程中发挥作用。

PROJECT SUMMARY 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 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; most 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.