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: 10152601
• 负责人: William J Snell
• 金额: $ 54.72万
• 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 2022-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152601
• 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; 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; 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.

我们将以双纤毛绿色披衣藻的受精作用为模型系统进行研究 纤毛信号传导和配子膜融合反应的保守细胞和分子机制。 当相反类型的衣原体配子混合在一起时， 纤毛上的互补粘附受体。粘附受体之间的相互作用（由 正配子中的SAG 1和负配子中的SAD 1）启动纤毛内的信号通路， 传递到细胞体和1）触发配子迅速（约5分钟）进行罢工 预先存在的纤毛粘附多肽从细胞体质膜重新分布到基底， 纤毛，然后运输到睫状膜上;和2）揭开并激活顶部定位 突起- -正负交配结构- -在两个纤毛之间的每个配子的细胞体上。 每个交配结构都带有一个配子特异性粘附蛋白，与纤毛上的粘附蛋白不同， 两个匹配结构的尖端在一起。交配结构的粘附迅速其次是脂双层 通过配子特异性广泛保守蛋白HAP 2的作用进行合并。融合后不久， 交配结构粘附分子和纤毛粘附受体被下调。在我们 通过对睫状体信号传导和蛋白运输的研究，我们发现睫状体粘连诱导的顶端 SAG 1多肽的定位依赖于细胞质中的单线态微管和 逆行鞭毛内转运（IFT）马达。另一方面，与目前的模式相反， SAG 1向纤毛的运输不需要顺行的IFT马达。最后，我们确定SAG 1 进入纤毛的运动是单向的。在下调过程中，SAG 1不会返回细胞体，但SAG 1的表达会降低。 蛋白质的全部预先存在的补体以纤毛外体的形式脱落到培养基中。我们 这些发现挑战了现存的，很大程度上未经测试的睫状膜蛋白运输模型， 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， 受体对中的一员，对正负配对结构之间的粘附至关重要。在哪方面 我们认为这是该领域的一个重大进展，我们发现融合必需的HAP 2蛋白是一种II类融合蛋白， 寨卡病毒与登革热病毒的融合蛋白属于同一家族。突变或免疫 对HAP 2“融合环”的干扰不干扰HAP 2在配对结构上的定位， 但使HAP 2在双层融合中失活。我们的发现使得详细的结构和功能- 基于真核配子融合的分子解剖。此外，它们还提供了一种可能性， 可用于干扰原生动物病原体的传播，包括 含有HAP 2的疟疾病原体疟原虫。