Gamete Membrane Adhesion and Fusion During Fertilization

受精过程中配子膜的粘附和融合

• 批准号: 9334510
• 负责人: William J Snell
• 金额: $ 15.68万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 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 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9334510
• 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; 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; Vascular Plant; Work; gene function; malaria transmission; malaria transmission-blocking vaccine; male; mutant; protein B; protein function; transcriptome; zygote

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.

项目摘要 男性和女配子在受精过程中形成合子的融合是生命中的决定性时刻 真核生物。尽管了解配子融合对于生殖健康至关重要，但我们尚不知道 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。我的 实验室使用双链球化的单细胞绿色藻类衣原体Reinhardtii作为模型系统来研究 受精。在施肥的第一阶段，Plus的鞭毛和负配子之间的粘附带来 它们在一起，还激活两者，以揭示专门用于融合的细胞膜位点。接下来， 融合质膜接触并立即融合。第一次 有机体，我们已经表明，两个膜的融合膜和合并的附着 在遗传上可以区分，并通过至少两个不同的整合膜蛋白进行。 pre 配子膜之间的融合附件受特定于物种的配子特异性蛋白质控制 FUS1和随后的膜合并取决于广泛保守的负配子特异性蛋白， hap2。 HAP2家庭成员出现在海绵中； Cnidarians；几只昆虫；最高的植物；和 许多毁灭性的致病生物，包括疟原虫。此外，我们还发现了 膜块的分子机制到多植物，表明FUS1和HAP2都经历了 衣原体膜块与一夫多妻制期间的快速，融合依赖性蛋白水解。这 衣原体系统有望允许我们使用配子融合的分子机理 知识和方法尚未用于其他系统。我们有良好的生物测定值可检测 并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 疟疾受精研究的意外影响。与我们的合作者一起，我们表明HAP2是 疟疾疟原配子融合和疟疾的传播至关重要，因此是新的质子 疟疾传输阻滞疫苗的靶标。了解至少一个生物，分子 配子膜融合反应期间发生的事件将对现场产生很大的影响 生殖生物学。这种知识将建立一个框架，以剖析 在其他生物体中的配子融合，用于避孕剂的发展和治疗不育症。这 此处介绍的研究策略的目的是测试HAP2用作融合蛋白的模型 在膜融合反应期间。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，我们将确定在膜融合过程中起作用的新蛋白质。