UNCONVENTIONAL MYOSINS IN RENAL FUNCTIONS

描述：(项目摘要)拟议的研究将继续研究以肌动蛋白为基础的细胞骨架在肾脏功能和疾病中的作用。我们将继续描述在肾脏中表达的多个基于肌动蛋白的马达的功能特性，重点是了解肌球蛋白在缺血性损伤中的作用。肾脏缺血导致肌动蛋白细胞骨架的快速变化和近端小管细胞膜极性的丧失。需要检验的假设是，一个或多个近端小管肌球蛋白依赖于ATP的机械力化学活动对于维持肾近端小管上皮细胞的极性以及在非致死性缺血损伤后恢复极性是必不可少的。为了了解这些作用，我们建议纯化和鉴定两个近端小管表达的肌球蛋白，肌球蛋白-VI和肌球蛋白-V11a。这些研究还将包括鉴定肌球蛋白尾部结合蛋白，这些蛋白可能将这些肌球蛋白与膜细胞骨架联系起来。我们还建议研究一系列肾脏肌球蛋白在肾脏损伤和恢复过程中的位置和细胞骨架的相关性。这些研究将包括免疫定位和生化提取技术，以评估肌球蛋白与膜细胞骨架的联系在损伤过程中是否受到调节。由于肾脏肌球蛋白的特征仍然不完整，我们建议使用基于聚合酶链式反应的筛查来鉴定新的肾脏肌球蛋白。具体地说，我们将寻找新的肾小球、远端小管和集合管肌球蛋白，并继续我们对近端小管肌球蛋白的分析。最后，我们建议评估肌球蛋白-VI(Snell‘s Waltzer)和肌球蛋白-VIIa(Shaker-1)突变的小鼠是否表现出肾功能障碍。这些突变的小鼠将被检测对肾脏损伤的敏感性以及对饮食压力的敏感性。通过分离和鉴定肾脏肌球蛋白，我们希望确定哪些肌球蛋白参与了在缺血损伤恢复过程中观察到的细胞骨架和细胞膜重排。

Description: (Abstract of the project) The proposed studies will continue the investigation of the role of the actin-based cytoskeleton in renal function and disease. We will continue to delineate the functional properties of the multiple actin-based motors expressed in the kidney with an emphasis on understanding the role of myosins in ischemic injury. Kidney ischemia leads to rapid changes in the actin cytoskeleton and a loss of membrane polarity in the proximal tubule cells. The hypothesis to be tested is that the ATP-dependent mechanochemical activity of one or more of the proximal tubule myosins is essential for maintenance of renal proximal tubule epithelial cell polarity, as well as for recovery of polarity following non-lethal ischemic injury. To understand these roles, we propose to purify and enzymatically characterize two proximal tubule expressed myosins, myosin-VI and myosin-Vlla. These studies will also include identification of myosin tail binding proteins that may link these myosins to the membrane cytoskeleton. We also propose to investigate the locations and cytoskeletal associations of an array of renal myosins during renal injury and recovery. These studies will include both immunolocation and biochemical extraction techniques to assess whether association of myosins with the membrane cytoskeleton is regulated during injury. As the characterization of renal myosins is still incomplete, we propose to identify, using a PCR-based screen, new renal myosins. Specifically, we will look for novel glomerular, distal tubule and collecting duct myosins as well as continue our analysis of proximal tubule myosins. Finally, we propose to assess whether mouse mutants with mutations in myosin-VI (Snell's waltzer) and myosin-VIIa (shaker-1) exhibit kidney dysfunction. These mutant mice will be assayed for sensitivity to renal injury as well as for susceptibility to dietary stress. By isolating and characterizing renal myosins we hope to identify which myosins are involved in the cytoskeletal and membrane rearrangements observed during recovery from ischemic injury.