Gamete Membrane Adhesion and Fusion During Fertilization

受精过程中配子膜的粘附和融合

• 批准号: 8538993
• 负责人: William J Snell
• 金额: $ 26.85万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538993
• 关键词: Adhesions; Binding; Biological; Biological Assay; Biological Models; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cnidaria; Collaborations; Complex; Contraceptive Agents; Culicidae; Development; Electrons; Essential Genes; Eukaryota; Event; Family member; Female; Fertility; Fertilization; Flagella; Gene Expression Profile; Genes; Germ Cells; Green Algae; Infertility; Insecta; Integral Membrane Protein; Knowledge; Laboratories; Life; Malaria; Mating Types; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microscopic; Modeling; Molecular; Molecular Genetics; Organism; PDAP2 Gene; Phase; Plasmodium; Polygamy; Porifera; Property; Proteins; Proteolysis; Reaction; Reproductive Biology; Reproductive Health; Research; Rodent; Site; Sperm-Ovum Interactions; Sterility; Structural Biochemistry; Structural Biologist; Structure; Surface; System; Testing; Time; Vaccines; Vascular Plant; Work; gene function; male; mutant; protein B; protein function; transmission process; zygote

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.

描述（由申请人提供）：在受精过程中，雄性和雌性配子融合形成合子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单个生物体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色衣藻作为模式系统来研究受精。在受精的第一阶段，正配子和负配子的鞭毛之间的粘附使它们聚集在一起，并激活两者以暴露专门用于融合的细胞膜位点。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。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FUS 1控制，随后的膜合并取决于广泛保守的负配子特异性蛋白HAP 2。HAP 2家族成员存在于海绵、刺胞动物、几种昆虫、高等植物和许多毁灭性的病原性原生生物中，包括疟原虫。此外，我们还发现了一个分子机制的膜块多精受精，表明FUS 1和HAP 2进行快速，融合依赖性蛋白水解在衣原体膜块多精受精。这个衣原体系统是准备让我们解剖配子融合的分子机制，使用知识和方法还没有为其他系统。我们有完善的生物测定来检测和量化配子相互作用中的每一步;我们有不育突变体在受精的几个步骤中被阻断;生物体很容易进行遗传和分子生物学操作;并且，我们可以制备足够的蛋白质用于生物化学和结构研究。我们对HAP 2的发现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HAP 2对于疟原虫配子融合和蚊子传播疟疾至关重要，因此是疟疾传播阻断疫苗的新主要靶点。对于至少一种生物，了解配子膜融合反应期间发生的分子事件将对生殖生物学领域产生重大影响。这些知识将建立一个框架，用于剖析其他生物体中配子融合的基本原理，并用于开发避孕药和治疗不孕症。本文提出的研究策略的目的是测试HAP 2在膜融合反应期间作为融合蛋白发挥作用的模型。我们将鉴定与HAP 2相互作用的蛋白质，我们将研究HAP 2的功能结构域，我们将鉴定在膜融合过程中起作用的新蛋白质。