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: 10797497
• 负责人: William J Snell
• 金额: $ 12.09万
• 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/10797497
• 关键词: 15 year old; Adenylate Cyclase; Adhesions; Biochemical; Biological Assay; Biological Models; Cell fusion; Cells; Cellular Assay; Chimeric Proteins; Chlamydomonas; Complex; Concentration Camps; Cyclic AMP; Fertilization; Flow Cytometry; Fluorescent Probes; Funding; Germ Cells; Green Algae; Huntingtin-Associated protein 1; Individual; Lasers; Measurement; Membrane Fusion; Membrane Proteins; Microscopic; Microscopy; Mutagenesis; PDAP2 Gene; Partner in relationship; Phenotype; Preparation; Production; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Reaction; Reagent; Research; Rest; Roentgen Rays; Sampling; Signal Transduction; Structure; System; Technology; Viral; instrument; monomer; mutant; protein function

We use the bi-ciliated, unicellular green alga Chlamydomonas as a model system to study conserved mechanisms of ciliary signaling and cell-cell fusion during fertilization. Ciliary adhesion between plus and minus gametes induces a rapid, ~15-fold increase in intracellular cAMP in the cell body that leads to activation of the cells in preparation for the gamete membrane fusion reaction. In our studies of ciliary signaling, we are examining the functions of the protein kinase GSPK, the adenylyl cyclase IMP4, and the protein phosphatase PP2A3 in the ciliary adhesion-induced increase in cAMP. One key assay in these studies is measurement of ciliary adhesion-induced cAMP production in gametes expressing wild type and mutant forms of the proteins. New fluorescent reagents are now being used in other systems to rapidly quantify cellular cAMP levels in live cells and we anticipate that they can be used with flow cytometry to quantify cAMP concentration in our cells. In studies of the mechanisms of gamete fusion, we showed that interaction between the membrane protein FUS1 on the mating structure of activated plus gametes with the membrane protein MAR1 on the mating structure of minus gametes leads to attachment of the two gametes at their mating structures. We have demonstrated that MAR1 is bifunctional. Not only does MAR1 interact with FUS1 during mating structure adhesion, but in resting minus gametes, MAR1 is biochemically and functionally associated on the minus mating structure with the conserved class II membrane fusion protein, HAP2. Unlike viral class II fusion proteins, however, whose fusogenic reconfiguration is triggered by low pH, the fusogenic reconfiguration of HAP2 is triggered by FUS1-MAR1 interactions. We and our collaborators have just determined the X-ray structure of the FUS1-MAR1 complex, showing that its is a heterotetramer composed of two MAR1 monomers and two FUS1 monomers. We will use structure-guided mutagenesis in combination with phenotypic analyses to identify the structural domains and specific residues in FUS1, MAR1, and HAP2 important both to maintain them in their prefusion forms and to regulate their function during the fusion reaction. We have just determined that a flow cytometry-based assay for cell-cell fusion is 20 times faster than our current microscopy-based assay and allows us to assess 30,000 cells/per sample compared to 300 cells/sample in our microscopic assay. The currently available flow cytometer is well past its prime, is unreliable, and lacks the laser technology needed for newly available fluorescent probes. Here, we are requesting funds towards the purchase of a new Becton Dickinson Symphony A1 Flow Cytometry system that we believe will substantively enhance the efficiency and thorougness of our research.

我们使用双纤维，单细胞绿色藻类衣原体作为研究保守的模型系统 受精过程中睫状信号传导和细胞融合的机制。加和之间的睫状粘合剂 减去配子会诱导细胞体内的细胞内营地迅速增加15倍，导致 细胞的激活为配子膜融合反应做准备。在我们的睫状研究中 信号传导，我们正在研究蛋白激酶GSPK，腺苷酸环化酶IMP4和蛋白质激酶的功能 纤毛粘附诱导的cAMP增加的蛋白质磷酸酶PP2A3。其中的一个关键测定法 研究是对睫状粘附诱导的cAMP产量的测量，表达野生类型和 蛋白质的突变形式。现在，新的荧光试剂正在其他系统中用于快速 量化活细胞中的细胞营地水平，我们预计它们可以与流式细胞仪一起使用 量化我们细胞中的cAMP浓度。在对配子融合机制的研究中，我们表明 膜蛋白FUS1之间在激活加配子的交配结构上的相互作用与 膜蛋白MAR1在负配子的交配结构上导致两个配子的附着 在其交配结构上。我们已经证明了MAR1是双功能的。 MAR1不仅互动 在交配结构粘附过程中使用FUS1，但是在静止的配子中，MAR1在生化上是生物化学和 与保守的II类膜融合蛋白的负相交结构在功能上相关， hap2。但是，与病毒II类融合蛋白不同，然而，其融合的重新配置是由低pH触发的 HAP2的融合重新配置是由FUS1-MAR1相互作用触发的。我们和我们的合作者 刚刚确定了FUS1-MAR1复合物的X射线结构，表明其是异驱量 由两个MAR1单体和两个FUS1单体组成。我们将在结构引导的诱变中使用 结合表型分析，以识别FUS1中的结构域和特定残基 MAR1和HAP2对将它们保持在预选形式并调节其功能很重要 在融合反应期间。我们刚刚确定基于流式细胞术的细胞融合测定是 比当前基于显微镜的测定快20倍，并允许我们评估30,000个单元/每个样品 与我们的微观测定中的300个细胞/样品相比。当前可用的流式细胞仪已经过去了 它的素数不可靠，缺乏新近可用的荧光探针所需的激光技术。这里， 我们要求资金购买新的Becton Dickinson Symphony A1流式细胞仪 我们认为将实质上提高我们研究的效率和胸部的系统。