Conserved mechanisms of ciliary signaling and cell-cell fusion

纤毛信号传导和细胞间融合的保守机制

• 批准号: 10707152
• 负责人: William J Snell
• 金额: $ 56.16万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707152
• 关键词: Adenylate Cyclase; Adhesions; Affect; Africa; Apical; Automobile Driving; Binding; Binding Proteins; Biochemical; Biological; Biological Models; Cell Adhesion Molecules; Cell Communication; Cell Fractionation; Cell Wall; Cell fusion; Cell membrane; Cell-Cell Adhesion; Cells; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Communication; Complement; Concentration Camps; Coupling; Culicidae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Cytoplasm; Dengue; Development; Disease; Dissection; Erinaceidae; Event; Evolution; Fertilization; Foundations; G-Protein-Coupled Receptors; Germ Cells; Green Algae; Hand; Homologous Gene; Human; Huntingtin-Associated protein 1; Image; Immunofluorescence Immunologic; Laboratory Study; Learning; Ligands; Link; Lipid Bilayers; Location; Malaria; Mating Types; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microtubules; Modeling; Molecular; Organelles; Organism; PDAP2 Gene; Partner in relationship; Pathway interactions; Peptides; Phosphorylation; Plasmodium; Process; Property; Protein Family; Protein Kinase; Protein S; Proteins; Reaction; Regulation; Rest; Signal Pathway; Signal Transduction; Site; Sperm-Ovum Interactions; Structure; System; Time; Vertebrates; Vesicle; Virus; ZIKA; adhesion receptor; body system; ciliopathy; erection; field study; malaria transmission; malaria transmission-blocking vaccine; male; patched protein; protein function; receptor; response; restraint; trafficking; transmission process

We propose to use fertilization in the green alga Chlamydomonas as a model system to investigate conserved but still poorly understood cellular and molecular mechanisms of ciliary signaling and the gamete membrane fusion reaction. When Chlamydomonas gametes of opposite mating types are mixed together they adhere to each other by complementary adhesion receptors on their cilia. Interactions between the adhesion receptors SAG1 on plus gametes and SAD1 on minus gametes initiate a signaling pathway that is transmitted to the cell body and triggers a rapid increase in cellular cAMP that activates the gamete for cell-cell fusion. Regulation of ciiliary cyclic nucleotides by ciliary receptor engagement has been conserved throughout evolution and in almost all ciliary signaling events in vertebrates, the primary response to engagement of ciliary receptor is a change in the concentration of cAMP within the organelles. Moreover, in most vertebrate ciliary systems, the mechanisms that transmit the increase in ciliary cAMP to a cellular response are unclear. We have discovered that a protein kinase, GSPK, in Chlamydomonas functions at a previously unrecognized step in ciliary signaling. GSPK is localized in the cytoplasm, not the cilia, yet the entire cellular complement of GSPK is phosphorylated within 1 minute after engagement of the ciliary adhesion receptors. Furthermore, GSPK phosphorylation is upstream of the cAMP increase and GSPK function is required for the rapid increase in cAMP. Our findings set the stage for new strategies to investigate cellular and molecular mechanisms that couple ciliary receptor engagement to a cytoplasmic response. We will use a combination of cell biological, biochemical, and imaging strategies to investigate the mechanisms underlying GSPK function. Gamete activation also activates erection of apically localized protrusions, the plus and minus mating structures, from the plasma membrane of each gamete that are the sites of membrane fusion during fertilization..We have made exciting advances in understanding conserved molecular mechanisms of the gamete membrane fusion reaction, and we now know more about cell- cell fusion in Chlamydomonas than is known in any other system. 1) We showed that bilayer merger of the two cells is driven by trimer formation of eukaryotic class II fusion protein, HAP2, a ancient protein required for fertilization in organisms across kingdoms. 2) We have now identified the separate cell-cell adhesion proteins s FUS1 and MAR1 at the fusion site of each cell. 3) We demonstrated that the adhesion protein MAR1 on minus gametes not only binds to FUS1 on plus gametes, but MAR1 is also biochemically and functionally linked to HAP2. 4) We showed that membrane adhesion mediated by FUS1-MAR1 interactions releases the restrained, prefusion form of HAP2, leading to the irreversible structural rearrangements into the fusion-driving HAP2 trimer. 5) In collaborative laboratory studies and field studies in Africa, we showed that targeting the conserved fusion loop of Plasmodium HAP2 inhibits the mosquito transmission of malaria. Our discoveries now make possible a detailed, structure- and function-based molecular dissection of ciliary signaling and gamete fusion..

我们建议在绿色藻层中使用施肥作为模型系统，以调查保守的 但仍然了解睫状信号传导和配子膜的细胞和分子机制知之甚少 融合反应。当相反交配类型的衣原体配子混合在一起时，他们会遵守 通过纤毛上的互补粘附受体彼此相互。粘附受体之间的相互作用 sag1 on Plus配子上的SAG1和减去配子的SAD1启动了传输到单元的信号传导途径 身体并触发细胞cAMP的迅速增加，从而激活配子进行细胞融合。调节 通过睫状受体互动的ciilariary环状核苷酸在整个进化过程中都保留了 脊椎动物中的所有睫状信号事件，对睫状受体参与的主要反应是变化 组织内的营地集中度。此外，在大多数脊椎动物睫状系统中，机制 将睫状营的增加传播到细胞反应尚不清楚。我们发现蛋白质 衣原体中的激酶GSPK在纤毛信号传导中以前无法识别的步骤起作用。 GSPK是 局部在细胞质中，而不是纤毛，但GSPK的整个细胞补体在1中磷酸化。 睫状粘附受体参与后分钟。此外，GSPK磷酸化是 营地迅速增加需要营地增加和GSPK功能。我们的发现为 研究将纤毛受体参与到一个细胞和分子机制的新策略 细胞质反应。我们将使用细胞生物学，生化和成像策略的组合来 研究GSPK函数的基础机制。配子激活还可以激活勃起 来自每个配子的质膜的局部突出，加号和减去交配结构 受精过程中的膜融合的位置是我们在理解方面取得了令人兴奋的进步 配子膜融合反应的保守分子机制，我们现在更多地了解细胞 衣原体中的细胞融合比任何其他系统中所知。 1）我们证明了两者的双层合并 细胞由真核II类融合蛋白的三聚体形成HAP2，这是一种古老的蛋白质 跨王国的生物体受精。 2）我们现在已经确定了单独的细胞 - 细胞粘附蛋白S 每个单元的融合位点的FUS1和MAR1。 3）我们证明了粘附蛋白MAR1对负蛋白 配子不仅与配子上的FUS1结合，而且MAR1在生化和功能上也链接到 hap2。 4）我们表明，FUS1-MAR1相互作用介导的膜粘附释放了约束， HAP2的预选形式，导致不可逆转的结构重排进入融合驱动HAP2三聚体。 5）在非洲的合作实验室研究和现场研究中，我们表明针对保守的融合 疟原虫HAP2的环抑制疟疾的蚊子传播。我们的发现现在使得 纤毛信号传导和配子融合的详细，结构和基于功能的分子解剖。