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.

我们将在双硅化的绿色藻类衣原体中使用受精作为研究模型系统 保守的睫状信号传导和配子膜融合反应的细胞和分子机制。 当相反类型的衣原体游戏混合在一起时，它们会彼此粘附在一起 在纤毛上完成了粘附受体。粘附受体之间的相互作用（由 sag1 in Plus Gamestes和sad1中的SAG1）在Cilia中启动了一个信号通路 传输到细胞体，1）迅速（〜5分钟）触发游戏 从细胞体质膜重新分布纤毛粘附多肽到底部 纤毛，然后贩运到睫状膜上； 2）揭示和激活顶端本地化 突起 - 两个纤毛之间每个配子的细胞体上的加号和减去交配结构。 每个交配结构都带有与纤毛上的粘合剂特异性粘合剂蛋白 两个交配结构的提示在一起。配合结构粘附迅速后面是脂质双层 通过配子特异性构造的蛋白质HAP2的作用合并。融合后不久 交配结构粘合分子和睫状粘合受体受体被下调。在我们的 睫状信号传导和蛋白质运输研究，我们发现睫状粘合剂诱导的根尖 SAG1多肽的定位取决于细胞质中的单个微管和作用 逆行内部运输（IFT）电动机。另一方面，与当前模型形成鲜明对比，动态 将SAG1贩运到纤毛中不需要正进的IFT电动机。最后，我们确定了SAG1 进入纤毛是单向的。在下调过程中，SAG1不会返回细胞体，而是 蛋白质的整个预先存在的蛋白质以纤毛etosomes的形式脱落成培养基。我们的 调查结果挑战现有的，在很大程度上未经测试的纤毛膜蛋白运输模型并设定了舞台 为了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，我们确定了缺失 受体对的成员对于加号和减去交配结构之间的广告至关重要。而且，在什么 我们认为这是该领域的重大进步，我们发现融合的HAP2蛋白是II类融合 与登革热和寨卡病毒融合蛋白同一家族中的蛋白质。突变或免疫学 干扰HAP2“ Fusion Loop”不会干扰HAP2与交配结构的定位， 但是使HAP2在双层融合中不活跃。我们的发现使得成为可能的详细，结构和功能 - 真核配子融合的基于分子解剖。此外，他们开放了策略的可能性 用于阻断病毒传播的用于干扰原生动物病原体的传播，包括 含HAP2的疟疾生物体，疟原虫。