STRUCTURE AND FUNCTION OF THE RENAL EPITHELIAL CELL MEMBRANE SKELETON

肾上皮细胞膜骨架的结构和功能

• 批准号: 6238936
• 负责人: MARK MOOSEKER
• 金额: $ 17.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238936
• 关键词: calcium; cell cell interaction; cell differentiation; cell membrane; cellular polarity; cytoskeleton; enzyme inhibitors; epithelium; immunocytochemistry; kidney; membrane activity; membrane potentials; membrane proteins; molecular cloning; myosins; nucleic acid sequence; phosphorylation; protein kinase; stimulant /agonist; tight junctions; tissue /cell culture; transfection

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.

拟议的研究将继续对分子的研究 肾脏膜骨架和紧密连接(TJ)的特征 上皮细胞。将追求两个具体的实验目标。 第一个是启动对分子和功能的研究 肌球蛋白家族肌动蛋白分子马达的特性 表达于近端小管上皮(PTE)细胞。分析将 从肌球蛋白-VI的特性开始，这是一种新发现的类别 与细胞器运输有关的非常规肌球蛋白 在PTE细胞中表达水平相对较高。一个全长的cDNA 猪肌球蛋白VI在细胞接触诱导分化过程中的编码 将对CL4细胞进行分析。肌球蛋白-VI将被提纯并进行生化 特色化的。有人提议用实验来鉴定“对接蛋白”。 它可能将肌球蛋白-VI连接到膜上。最后，为了努力 确定肌球蛋白-VI在PTE细胞中的功能，稳定的CL4株将 产生过表达肌球蛋白VI的尾巴截断或缺乏肌球蛋白VI 由于反义转录表达。我们的第二个目标是调查 肾脏TJ调节的分子机制。这一结构及其 足细胞变体，裂隙隔膜，形成旁细胞屏障和 维持细胞的极性。在正常情况下，屏障属性受到调节 并在肾脏病理中发生改变。有证据表明蛋白质 蛋白激酶调节肾脏的组装、拆解和屏障特性 TJS。我们将测试是否调节中心是磷酸化的 TJ特异性蛋白。MDCK细胞，一个肾小管模型，缺乏 低钙培养时细胞间的连接和极性； 跨单分子层电学的连接组装与建立 抗性是通过提高细胞外钙离子浓度来诱导的。磷酸化 四种TJ蛋白的状态将与它们的变化相关 由单层电学监测的TJ组装过程中的定位 在这种钙离子交换过程中的阻力。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 将会被检查。我们最近观察到，不同亚型的 TJ蛋白ZO-1在肾小管上皮细胞和肾小管上皮细胞的表达 内皮细胞和足细胞连接。这种分布与 存在结构和功能上的差异。试点研究将审查 如果ZO-1亚型在人类肾脏疾病中表达异常 有助于改变肾小球和肾小管屏障功能。