Gamete Membrane Adhesion and Fusion During Fertilization

项目总结 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。尽管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也不需要分子步骤.我的 实验室以双鞭毛单细胞绿藻莱茵衣藻为模型系统进行研究 受精。在受精的第一阶段，正负配子鞭毛之间的粘连带来 它们结合在一起，也都被激活，暴露出专门用于融合的细胞膜部位。接下来， 融合质膜紧密接触并立即融合。这是有史以来第一次 生物体，我们现在已经证明了融合基膜的附着和两种膜的合并 在基因上是可区分的，至少由两种不同的整膜蛋白执行。Pre- 配子膜之间的融合附着由物种特异的加配子特异的蛋白质控制 FUS1，以及随后的膜合并依赖于广泛保守的负配子特异蛋白， HAP2。HAP2家族成员存在于海绵、蛇形目动物、几种昆虫、大多数高等植物以及 许多毁灭性的致病原生生物，包括疟原虫。此外，我们还发现了一种 膜阻断多精母细胞的分子机制，证明FUS1和HAP2都经历了 在衣藻菌膜阻断到一夫多妻制的过程中，依赖于融合的快速蛋白质分解。这 衣藻系统准备好让我们用来剖析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来检测 并量化配子相互作用中的每一步；我们在受精的几个步骤中阻止了不育突变； 生物体很容易受到遗传和分子生物学操作的影响；而且，我们可以准备 足够进行生物化学和结构研究的蛋白质数量。我们发现的HAP2已经有了 在疟疾施肥研究中产生了意想不到的影响。与我们的合作者一起，我们展示了HAP2是 对疟原虫配子融合和蚊子传播疟疾是必不可少的，因此是一种新的素材 疟疾传播阻断疫苗的目标。对于至少一个有机体来说，理解分子 配子膜融合反应过程中发生的事件将对 生殖生物学。这些知识将建立一个框架，用于剖析 配子在其他生物体中的融合，用于避孕药具的开发和治疗不孕不育。这个 这里提出的研究策略的目的是测试HAP2作为融合蛋白发挥作用的模型 在膜融合反应过程中。我们将鉴定与HAP2相互作用的蛋白质，我们将研究 HAP2的功能结构域，我们将鉴定在膜融合过程中发挥功能的新蛋白。

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.