Membrane protein localization and function during ciliary signaling and cell-cell fusion

纤毛信号传导和细胞-细胞融合过程中膜蛋白的定位和功能

• 批准号: 9277022
• 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: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9277022
• 关键词: Adhesions; Apical; Binding; Biological Models; Cell Adhesion Molecules; Cell fusion; Cell membrane; Cells; Chimeric Proteins; Chlamydomonas; Cilia; Complement; Cytoplasm; Dengue Virus; Dissection; Down-Regulation; Employee Strikes; Family; Fertilization; Foundations; Germ Cells; Green Algae; Huntingtin-Associated protein 1; Immunologics; Lipid Bilayers; Malaria; Membrane; Membrane Fusion; Membrane Proteins; Microtubules; Modeling; Molecular; Motor; Movement; Mutation; Organism; Partner in relationship; Plasmodium; Property; Proteins; Reaction; Regulation; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Ursidae Family; Vitronectin; Zika Virus; adhesion receptor; base; member; pathogen; polypeptide; protein transport; receptor; trafficking; transmission process; viral transmission

We will use fertilization in the bi-ciliated green alga Chlamydomonas as a model system to investigate conserved cellular and molecular mechanisms of ciliary signaling and the gamete membrane fusion reaction. When Chlamydomonas gametes of opposite types are mixed together they adhere to each other by complementary adhesion receptors on their cilia. Interaction between the adhesion receptors (encoded by SAG1 in plus gametes and SAD1 in minus gametes) initiates a signaling pathway within the cilia that is transmitted to the cell body and 1) triggers the gametes rapidly (~ 5 minutes) to undergo a striking redistribution of pre-existing ciliary adhesion polypeptides from the cell body plasma membrane to the base of the cilium, followed by trafficking onto the ciliary membrane; and 2) uncovers and activates apically localized protrusions - - the plus and minus mating structures - - on the cell body of each gamete between the two cilia. Each mating structure bears a gamete-specific adhesion protein, distinct from those on the cilia, that bind the tips of the two mating structures together. Mating structure adhesion is rapidly followed by lipid bilayer merger through the action of the gamete-specific broadly conserved protein, HAP2. Soon after fusion, the mating structure adhesion molecules and the ciliary adhesion receptors receptors are down-regulated. In our ciliary signaling and protein trafficking studies, we have found that ciliary adhesion-induced apical localization of SAG1 polypeptide depends on singlet microtubules in the cytoplasm and action of the retrograde intraflagellar transport (IFT) motor. On the other hand, contrary to current models, dynamic trafficking of SAG1 into cilia does not require the anterograde IFT motor. Finally, we determined that SAG1 movement into cilia is uni-directional. During down-regulation, SAG1 does not return to the cell body, but the entire pre-existing complement of the protein is shed into the medium in the form of ciliary ectosomes. Our findings challenge existing, largely untested models of ciliary membrane protein trafficking and set the stage for new strategies to investigate cellular and molecular mechanisms of the dynamic regulation of the protein composition of cilia. In our studies of the gamete membrane fusion reaction, we have identified the missing member of a receptor pair essential for adhesion between the plus and minus mating structures. And, in what we feel is a major advance in the field, we discovered that the fusion-essential HAP2 protein is a Class II fusion protein in the same family as Dengue and Zika virus fusion proteins. Mutational or immunological interference with the HAP2 “fusion loop” does not interfere with HAP2 localization to the mating structure, but renders HAP2 inactive in bilayer fusion. Our discoveries make possible a detailed, structure- and function- based molecular dissection of eukaryotic gamete fusion. Furthermore they open the possibility that strategies used to block viral transmission can be used to interfere with transmission of protozoan pathogens, including the HAP2-containing malaria organism, Plasmodium.

我们将以双纤毛绿藻衣藻中的受精为模式系统进行研究 纤毛信号和配子膜融合反应的保守细胞和分子机制。 当相反类型的衣藻配子混合在一起时，它们通过 纤毛上的互补黏附受体。黏附受体之间的相互作用(由 正配子中的SAG1和负配子中的SAD1)启动纤毛内的信号通路，即 传递到细胞体，1)迅速(~5分钟)触发配子经历打击 原存在的纤毛黏附多肽从细胞体质膜到基质的重新分布 纤毛，然后运输到睫状膜上；2)发现并激活顶端定位 在两个纤毛之间的每个配子的细胞体上，突起--正负交配结构。 每个交配结构都有一个配子特异的黏附蛋白，与纤毛上的不同，它结合 两个交配结构的尖端在一起。配对结构的粘连很快就会被脂类双层所跟随 通过配子特异的广泛保守的蛋白质HAP2的作用进行合并。核聚变后不久， 交配结构黏附分子和纤毛黏附受体下调。在我们的 纤毛信号和蛋白转运的研究，我们发现纤毛粘连诱导心尖部 SAG1多肽的定位依赖于细胞质中的单个微管和 逆行鞭毛内转运(IFT)运动。另一方面，与当前的模型相反，动态的 将SAG1运送到纤毛不需要顺行IFT马达。最后，我们确定SAG1 进入纤毛的运动是单向的。在下调过程中，SAG1不会返回到细胞体，但 整个先前存在的蛋白质补体以纤毛外体的形式被释放到介质中。我们的 这些发现挑战了现有的、基本上未经测试的睫状膜蛋白运输模型，并奠定了基础 寻找研究蛋白质动态调节的细胞和分子机制的新策略 纤毛的组成。在我们对配子膜融合反应的研究中，我们已经确定了缺失的 对正负交配结构之间的粘合至关重要的受体对的成员。而且，在什么方面 我们觉得这是该领域的一大进步，我们发现融合必需的HAP2蛋白是II类融合 与登革热和寨卡病毒融合蛋白属于同一家族的蛋白质。突变或免疫性 对HAP2“融合环”的干扰不会干扰HAP2对配对结构的定位， 但使HAP2在双层融合中不起作用。我们的发现使详细的结构和功能成为可能- 基于真核配子融合的分子解剖。此外，它们打开了一种可能性，即战略 用于阻断病毒传播可用于干扰原生动物病原体的传播，包括 含有HAP2的疟疾有机体--疟原虫。