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..

我们建议使用施肥在绿色披衣作为模式系统，以调查保护 但对纤毛信号传导和配子膜的细胞和分子机制仍知之甚少 聚变反应当相反交配类型的衣原体配子混合在一起时， 通过纤毛上的互补粘附受体相互作用。粘附受体之间的相互作用 正配子上的SAG 1和负配子上的SAD 1启动传递到细胞的信号通路 在体内，它会触发细胞内cAMP的快速增加，从而激活配子进行细胞-细胞融合。调控 睫状环核苷酸通过睫状受体接合在整个进化过程中是保守的， 在脊椎动物的所有纤毛信号事件中，对纤毛受体参与的主要反应是 细胞器内cAMP的浓度。此外，在大多数脊椎动物的纤毛系统中， 将睫状体cAMP的增加传递到细胞反应的机制尚不清楚。我们发现一种蛋白质 衣原体中的GSPK在纤毛信号传导中的一个以前未被认识的步骤起作用。GSPK是 定位于细胞质中，而不是纤毛，但整个细胞补体的GSPK磷酸化在1 在睫状体粘附受体接合后10分钟。此外，GSPK磷酸化是 cAMP增加和GSPK功能是cAMP快速增加所必需的。我们的发现为 研究睫状体受体参与与一种新的细胞和分子机制的新策略 胞质反应我们将使用细胞生物学、生物化学和成像策略的组合， 研究GSPK功能的潜在机制。配子激活也激活顶端的勃起 每个配子的质膜上的局部突起，即正负交配结构， 是受精过程中膜融合的部位。我们已经在理解 配子膜融合反应的保守分子机制，我们现在知道更多关于细胞- 衣原体细胞融合比任何其他系统中已知的。1)我们发现， 细胞是由真核II类融合蛋白HAP 2的三聚体形成驱动的，HAP 2是一种古老的蛋白质， 在生物界中的受精作用。2)我们现在已经鉴定了不同的细胞-细胞粘附蛋白 FUS 1和MAR 1在每个细胞的融合位点。3)我们证明了粘附蛋白MAR 1在负性细胞上的表达， 配子不仅与正配子上的FUS 1结合，而且MAR 1也在生物化学和功能上与 HAP2。4)我们发现，由FUS 1-MAR 1相互作用介导的膜粘附释放了受抑制的， 融合前形式的HAP 2，导致不可逆的结构重排为融合驱动的HAP 2三聚体。 5)在非洲的合作实验室研究和实地研究中，我们发现， 疟原虫HAP 2环抑制蚊子传播疟疾。我们的发现使得 详细的，基于结构和功能的纤毛信号传导和配子融合的分子解剖。