Hornomal Modification of Communication in Cultured Animal Cells (Biology)

Mary Lee Ledbetter博士之前的工作是用简单的方法来测量培养的成纤维细胞或上皮细胞通过间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瓦巴因处理细胞来抑制。当肾源性上皮细胞用类固醇激素醛固酮处理时，它们的瓦巴因抑制作用降低。最近的研究表明，钙的变化介导了瓦巴因的作用，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，以确定瓦巴因和醛固酮是否也改变连接蛋白的表达和/或分布，连接蛋白是一种间隙连接蛋白，在合作细胞中。实验方法包括：用免疫荧光染色法在完整细胞中定位连接蛋白；细胞提取物中连接蛋白特异性RNA和蛋白的表达及蛋白的电泳迁移率；以及通过染料注射测量的功能性通讯的快速变化。这些研究不仅将为了解瓦巴因、醛固酮与细胞通讯之间的相互作用提供更完整的基础，也将为今后研究间隙连接通讯的结构-功能关系提供更好的工具。互动活动包括参与斯克里普斯研究所一年级研究生的核心课程。这包括领导讨论部分，设计和实施实验体验，以及在课程中偶尔提供讲座。

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.