Gamete Membrane Adhesion and Fusion During Fertilization

受精过程中配子膜的粘附和融合

• 批准号: 8725175
• 负责人: William J Snell
• 金额: $ 27.83万
• 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 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725175
• 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）：男女配子在受精过程中形成合子的融合是真核生物生命中的决定时刻。尽管了解配子融合对于生殖健康至关重要，但即使是单个生物体，我们尚不知道分子也不知道配子膜融合所需的分子步骤。我的实验室使用双链球化的单细胞绿色藻类衣原体Reinhardtii作为研究施肥的模型系统。在施肥的第一阶段，Plus和负配子的鞭毛之间的粘附将它们融合在一起，并激活两者以暴露用于融合的细胞膜位点。接下来，融合质膜接触并立即融合。现在，在任何生物体中，我们都首次表明，两个膜的融合膜和合并的附着在遗传上是可以区分的，并且至少由两个不同的整合膜蛋白进行。配子膜之间的预融合附着受物种特异性加角色特异性蛋白FUS1的控制，随后的膜合并取决于广泛保守的，负配子特异性的蛋白质HAP2。 HAP2家庭成员出现在海绵中； Cnidarians；几只昆虫；较高的植物；以及许多毁灭性的致病生物，包括疟原虫。此外，我们还发现了一种分子机制，用于膜块到多植物，表明FUS1和HAP2在衣原体膜块中均经历了快速的，融合依赖性的蛋白水解。这种衣原体系统有望让我们使用尚不可用于其他系统的知识和方法来剖析配子融合的分子机制。我们有良好的生物测定，可以检测和量化配子相互作用的每个步骤。我们的无菌突变体在受精的几个步骤中被阻塞。该生物很容易受到遗传和分子生​​物学操纵的态度。而且，我们可以准备足以用于生物化学和结构研究的蛋白质。我们对HAP2的发现已经在疟疾受精研究中产生了意外的影响。与我们的合作者一起，我们表明HAP2对于疟疾疟原配子融合和疟疾传播至关重要，因此是疟疾传播阻断疫苗的新主要目标。了解至少一个生物，在配子膜融合反应中发生的分子事件将对生殖生物学领域产生很大影响。这种知识将建立一个框架，以剖析其他生物体中配子融合的基本原理，并开发避孕药具和治疗不育。这里提出的研究策略的目的是测试HAP2在膜融合反应过程中起融合蛋白的模型。我们将确定与HAP2相互作用的蛋白质，我们将研究HAP2的功能域，并将确定在膜融合过程中起作用的新蛋白质。