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.

我们使用双纤毛单细胞绿色衣原体作为模型系统，研究保守的 受精过程中纤毛信号传导和细胞-细胞融合的机制。睫状体粘连， 负配子诱导细胞体中细胞内cAMP快速增加~15倍，导致 激活细胞以准备配子膜融合反应。在我们对纤毛的研究中， 信号，我们正在研究蛋白激酶GSPK，腺苷酸环化酶IMP 4， 蛋白磷酸酶PP 2A 3在睫状体粘连诱导的cAMP增加中的作用。其中一项关键检测 研究是在表达野生型和野生型的配子中测量睫状体粘连诱导的cAMP产生， 蛋白质的突变形式。新的荧光试剂现在正用于其他系统， 定量活细胞中的细胞cAMP水平，我们预计它们可以与流式细胞术一起使用， 定量我们细胞中的cAMP浓度。在配子融合机制的研究中，我们发现， 膜蛋白FUS 1对激活正配子交配结构的影响与 负配子的交配结构上的膜蛋白MAR 1导致两个配子的附着 它们的交配结构。我们已经证明MAR 1是双功能的。MAR 1不仅与 与FUS 1在交配结构粘附，但在休息负配子，MAR 1是生化和 在负交配结构上与保守的II类膜融合蛋白功能相关， HAP2。然而，与病毒II类融合蛋白不同，其融合重构是由低pH触发的， HAP 2的融合重组由FUS 1-MAR 1相互作用触发。我们和我们的合作者 我刚刚确定了FUS 1-MAR 1复合物的X射线结构，表明它是一个异源四聚体 由两个MAR 1单体和两个FUS 1单体组成。我们将使用结构导向诱变技术， 结合表型分析以鉴定FUS 1中的结构域和特定残基， MAR 1和HAP 2对维持它们在融合前的形式和调节它们的功能都很重要 在聚变反应中。我们刚刚确定了一种基于流式细胞术的细胞-细胞融合测定法， 比我们目前基于显微镜的分析快20倍，允许我们评估30，000个细胞/每份样品 而在我们的显微分析中是300个细胞/样品。目前可用的流式细胞仪已经过时 它的prime是不可靠的，并且缺乏新可用的荧光探针所需的激光技术。在这里， 我们正在申请购买新的Becton Dickinson Symphony A1流式细胞仪的资金 我们相信这一系统将大大提高我们研究的效率和准确性。