STRUCTURE AND FUNCTION OF THE RENAL EPITHELIAL CELL MEMBRANE SKELETON

拟议的研究将继续调查分子 肾细胞膜骨架和紧密连接（TJs）的特征 上皮细胞 将追求两个具体的实验目标。 第一个将是启动分子和功能研究， 肌动蛋白基分子马达肌球蛋白家族的表征 在近端小管上皮（PTE）细胞中表达。 分析将 开始与肌球蛋白-VI，一个新发现的类的特性 与细胞器运输有关的非常规肌球蛋白， 在PTE细胞中以相对高的水平表达。 全长cDNA 在细胞接触诱导的分化过程中编码猪肌球蛋白-VI， 将分析CL 4细胞。 肌球蛋白-VI将被纯化， 表征了 实验提出了识别“对接蛋白” 其可以将肌球蛋白-VI连接到膜上。 最后，为了 为了确定PTE细胞中肌球蛋白-VI的功能，稳定的CL 4系将 产生过表达肌球蛋白VI尾部截短或缺乏肌球蛋白VI 这是由于反义转录本的表达。 我们的第二个目标是研究 肾TJ调节的分子机制。 这种结构及其 足细胞变体，狭缝隔膜，形成细胞旁屏障， 维持细胞极性。 在正常情况下， 在肾脏病理学中改变。 证据表明蛋白质 激酶调节肾细胞的组装、拆卸和屏障特性 TJ 我们将测试调节的位点是否是磷酸化的 TJ特异性蛋白质。 MDCK细胞，肾小管模型，缺乏 低Ca++培养时细胞间连接和极性; 跨膜电连接组装和建立 通过提高细胞外Ca++诱导抗性。 磷酸化 四种TJ蛋白的状态将与它们的 在TJ组装过程中的定位，如通过单层电 在这个Ca++开关的电阻。 蛋白激酶抑制剂的作用 和激动剂对磷酸化、结构和生理参数的影响 将被审查。 我们最近观察到， TJ蛋白ZO-1在肾小管上皮细胞中表达， 内皮细胞和足细胞连接。 这种分布与 结构和功能上的差异。 试点研究将审查 如果ZO-1同种型表达在人肾脏疾病中异常， 有助于改变肾小球和肾小管屏障功能。

The proposed studies will continue the investigation of the molecular characterization of membrane skeleton and tight junctions (TJs) of renal epithelial cells. Two specific experimental objectives will be pursued. The first will be to initiate studies on the molecular and functional characterization of the myosin family of actin-based molecular motors expressed in the proximal tubule epithelial (PTE) cells. Analysis will begin with the characterization of myosin-VI, a newly discovered class of unconventional myosin implicated in organelle transport that is expressed in relatively high levels in the PTE cell. A full length cDNA encoding pig myosins-VI during cell-contact induced differentiation of CL4 cells will be analyzed. Myosin-VI will be purified and biochemically characterized. Experiments are proposed to identify "docking proteins" which may link myosin-VI to the membrane. Finally, in an effort to determine the function of myosin-VI in PTE cells, stable CL4 lines will be created which overexpress myosin VI tail truncates or lack myosin-VI due to anti-sense transcript expression. Our second aim investigates molecular mechanisms of renal TJ regulation. This structure and its podocyte variant, the slit diaphragm, form the paracellular barrier and maintains cell polarity. Barrier properties are regulated during normal physiology and are altered in renal pathology. Evidence suggests protein kinases regulate assembly, disassembly and barrier properties of renal TJs. We will test whether the locus of regulation is phosphorylation of TJ-specific proteins. MDCK cells, a renal tubular model, lack intercellular junctions and polarity when cultured at low Ca++; junctional assembly and establishment of transmonolayer electrical resistance is induced by raising extracellular Ca++. The phosphorylation sate of four TJ proteins will be correlated with changes in their localization during TJ assembly, as monitored by monolayer electrical resistance during this Ca++ switch. Effects of protein kinase inhibitors and agonists on phosphorylation, structural and physiologic parameters will be examined. We recently observed that different isoforms of the TJ protein ZO-1 are expressed in renal tubular epithelial cells vs. renal endothelial cells and podocyte junctions. This distribution correlates with structural and functional differences. Pilot studies will examine if ZO-1 isoform expression is aberrant in human renal diseases and contributes to altered glomerular and tubular barrier function.