Hornomal Modification of Communication in Cultured Animal Cells (Biology)

玛丽·李·莱德贝特博士之前的工作使用简单的方法来测量培养的成纤维细胞或上皮细胞通过缝隙连接共享小细胞质分子的能力。这种合作可以通过用钠泵抑制剂哇巴因处理细胞来抑制。当肾上皮细胞用类固醇激素醛固酮处理时，哇巴因抑制作用减弱。最近的研究表明，钙离子的变化介导哇巴因的作用，而醛固酮的作用可能涉及一种不依赖于细胞质矿物皮质激素受体的不寻常机制。将进行测试，以确定哇巴因和醛固酮是否也改变合作细胞中缝隙连接蛋白的表达和/或分布。实验方法包括：通过免疫荧光染色在完整细胞中定位连接蛋白；在细胞提取物中表达连接蛋白特定的RNA以及蛋白质和蛋白质的电泳率；以及通过染料注射测量功能通讯的快速变化。这些研究不仅将为理解哇巴因、醛固酮和细胞通讯之间的相互作用提供更完整的基础，也将为今后的工作提供更好的工具来研究缝隙连接通讯中的结构-功能关系。互动活动包括参加斯克里普斯研究所一年级研究生的核心课程。这包括引导讨论部分，设计和实施实验经验，并在课程中提供不定期的讲课。

Dr. Mary Lee Ledbetter's previous work has used simple assays to measure the ability of cultured fibroblastic or epithelial cells to share small cytoplasmic molecules through gap junctions. This cooperation can be inhibited by treatment of the cells with the sodium-pump inhibitor ouabain. When kidney-derived epithelial cells are treated with the steroid hormone aldosterone, their ouabain inhibition is reduced. Recent work has suggested that calcium changes mediate the effects of ouabain and that aldosterone's action may involve an unusual mechanism independent of the cytoplasmic mineralocorticoid receptor. Tests will be carried out to determine whether ouabain and aldosterone also modify the expression an/or distribution of connexins, the gap junction protein, in cooperating cells. Experimental approaches include: connexin protein localization by immunofluorescent staining in intact cells; expression of connexin - specific RNA and protein and protein electrophoretic mobility in cell extracts; and rapid changes in functional communication, measured by dye injection. These studies will not only provide a more complete basis for understanding the interaction between ouabain, aldosterone and cell communication, they will also provide improved tools to study structure-function relationships in gap junction communication for future work. Interactive activities include participation in the core course for first-year graduate students at The Scripps Research Institute. This involves leading discussion sections, designing and implementing laboratory experiences, and offering occasional lectures in the course.