THE CELL BIOLOGY OF ASCIDIAN SPERM ACTIVATION

该项目的目标是增加参与的少数民族人数 在研究解决生育的生物医学问题， 受精，跨膜信号传导，以及细胞内运动， 细胞器通过研究激活的海鞘精子在细胞和 生化水平。 30%的学生谁在我的工作' 在过去的十年里，实验室一直是少数民族。这些学生中的许多 进入卫生专业，但很少有人继续从事生物医学 research. 我实验室的学生参与MBRS项目 将增加他们接触生物医学研究作为一个可行的职业 选项.这些学生将研究受精的早期事件 包括精卵结合、精子激活以及随后精子 卵膜的渗透，最终导致融合的过程 精子和卵子质膜。 精子的细胞机制 激活和渗透，由精卵结合触发，是 这个项目的焦点。 据推测， 必须将结合与激活联系起来， 产生力的细胞骨架元件的重排 激活线粒体迁移。 研究跨膜 信号传导连接需要用激活剂或阻断剂装载细胞 间接检测是否存在 多磷脂酰肌醇连接机制。 直接证据将被收集 通过对该途径的一些蛋白质成员的生化分析， 测量他们行为的产物的外观，并通过研究 它们在抗体存在下的行为， 干扰他们。这种多磷脂依赖性调节 选择这一途径是因为，在某些细胞中， 细胞内pH和钙，这两者都是海鞘的一部分， 精子激活 研究线粒体运动需要探测 参与细胞骨架的生化性质和细胞定位 proteins.特定细胞骨架蛋白的存在与运动性 马达将通过聚丙烯酰胺凝胶电泳建立， 免疫印迹。 这些蛋白质的细胞定位将被研究 通过间接免疫荧光和免疫电子显微镜。

The goal of this project is to increase the number of minorities involved in research addressing the biomedical problems of fertility and fertilization, signaling across membrane, and intracellular movement of organelles via studies of activated ascidian sperm at the cellular and biochemical levels. Thirty percent of the students who have worked in my' lab over the past ten years have been minorities. Many of these students have gone into health professions, but few have continued in biomedical research. The participation of students in my lab in the MBRS program would increase their exposure to biomedical research as a viable career option. These students would be studying the early events of fertilization include sperm-egg binding, sperm activation and, subsequently, sperm penetration of egg vestments, processes which ultimately lead to fusion of sperm and egg plasma membranes. The cellular mechanisms of sperm activation and penetration, as triggered by sperm-egg binding, are the foci of this project. It is hypothesized that signaling events which cross the membrane must connect binding to activation and that rearrangement of force-producing cytoskeletal elements must connect activation to mitochondrial migration. Investigating the transmembrane signaling connection requires loading of cells with activators or blockers to indirectly test for the presence of the components of a polyphosphainositide-linked mechanism. Direct evidence will be gathered by biochemical analysis of some of the protein members of the pathway, by measuring the appearance of the products of their actions, and by studying their behavior in the presence of antibodies which either identify or interfere with them. This polyphosphainositide-dependent regulatory pathway was chosen because, in some cells, it is known to elevate intracellular pH and calcium, both of which occur as part of ascidian sperm activation. Studying mitochondrial movement requires probing the biochemical nature and cellular location of participating cytoskeletal proteins. The presence of specific cytoskeletal proteins and motility motors will be established by polyacrylamide gel electrophoresis and immunoblotting. The cellular location of these proteins will be studied by indirect immunofluorescence and immunoelectron microscopy.