Apical intermediate filament (IF) mediated post ischemia recovery in epithelia

描述（由申请人提供）：缺血/再灌注（I/R）损伤导致肾近端小管细胞中微管（MT）和F-肌动蛋白的损失。使用轻度缺血模型（30分钟单侧肾血管钳夹），我们已经表明，这些细胞骨架元素在I/R后第3天大部分重新聚合，但质膜的极化仅在第5-7天变得正常，这与肾功能参数的正常化平行。在I/R后恢复的晚期阶段期间观察到异常MT极性，使我们假设微管组织中心（MTOC）在那时重新分布，因为MTOC组分GCP 6在位于aa 1395和1399之间的调节盒中磷酸化，GCP 6介导MTOC与中间丝的附着。因此，我提出以下具体目标来检验这一假设：1。确定GCP 6在MTOC附着于简单上皮细胞顶端结构域下的角蛋白中间丝中的作用。1.1使用缺失突变体确定角蛋白8/19的GCP 6结合结构域。1.2使用相同的方法定义角蛋白8中GCP 6的结合结构域。1.3过量表达上述突变体以鉴定对MTOC定位的显性-负性效应。2.表征已知阻断MTOC与角蛋白附着的激酶对GCP 6的磷酸化作用。2.1鉴定p34 cdc和plkl磷酸化位点。2.2在该位点获得GCP 6的S->D和S->A突变体，并验证角蛋白8结合和对p34 cc/c2信号传导的响应的变化。2.3过度表达这些突变体，并分析它们的定位和组织MT正常结构的能力。3.鉴定I/R后体内修饰的GCP 6的磷酸化位点，其解释了近端小管细胞中I/R后3-5天期间MTOC的脱离，并确定MTOC混乱对上皮极性的影响。3.1使用特异性抗磷酸肽抗体测定缺血后肾脏中GCP 6-S1396磷酸化的动力学，定位磷酸-81396 GCP 6，并测定体内I/R后GSK 3特异性阻断剂对MTOC分布的影响。3.2获得en 3.1研究的磷酸化位点的S->D突变体（可能是S1397附近的GSK-3位点），并验证缺乏与角蛋白的结合以及对体内MTOC定位的影响。3.3评估阻断MTOC与顶端IP附着后顶端质膜蛋白的去极化、载体膜递送和紧密连接功能的变化。该项目的长期目标是表征负责I/R后恢复后期MTOC和质膜蛋白去极化的信号通路，并设计干预措施以恢复它并改善I/R后的恢复时间。非正式声明：急性肾衰竭影响5%的住院患者，是需要肾移植的主要原因。在这个项目中，我们分析了在恢复的后半段组织细胞骨架的结构和信号传导，以及可能缩短肾衰竭期并改善其恢复的方法。这些概念也可以应用于肾移植的恢复，也遭受暂时缺乏血液供应的后果。

DESCRIPTION (provided by applicant): The ischemia/reperfusion (I/R) injury results in loss of microtubules (MTs) and F-actin in kidney proximal tubule cells. Using a model of mild ischemia (30-minute unilateral clamp of renal vessels) we have shown that those cytoskeletal elements are mostly repolymerized by day 3 after I/R, but polarization of the plasma membrane becomes normal only by day 5-7, which parallels the normalization of kidney functional parameters. The observation of abnormal MT polarity during the late phase of the recovery after I/R, lead us to hypothesize that Microtubule-Organizing Centers (MTOCs) are redistributed at that time because an MTOC component, GCP6, which mediates attachment of MTOCs to intermediate filaments, is phosphorylated in a regulatory cassette located between aa 1395 and 1399. Therefore, I propose the following specific aims to test this hypothesis: 1. Determine the role of GCP6 in the attachment of MTOCs to keratin intermediate filaments under the apical domain of simple epithelial cells. 1.1 Determine the binding domains in GCP6 for keratins 8/19 using deletion mutants. 1.2 Define the binding domains in keratin 8 for GCP6 using the same method. 1.3 Over-express the mutants mentioned above to identify dominant-negative effects on the localization of MTOCs. 2. Characterize the phosphorylation of GCP6 by kinases known to block the attachment of MTOCs to keratins. 2.1 Identify the p34cdc and plkl phosphorylation sites. 2.2 Obtain S->D and S->A mutants of GCP6 in that site and verify changes in keratin 8 binding and response to p34cc/c2 signaling. 2.3 Overexpress those mutants and analyze their localization and ability to organize the normal architecture of MTs. 3. Identify phosphorylation sites of GCP6 modified in vivo after I/R that account detachment of MTOCs during days 3-5 after I/R in proximal tubule cells and determine the consequences of a disarray of MTOCs on epithelial polarity. 3.1 Determine the kinetics of phosphorylation of GCP6-S1396 in post-ischemic kidney using a specific anti-phosphopeptide antibody, localize phospho-81396 GCP6, and determine the effect of specific blockers of GSK3 on MTOC distribution after I/R in vivo. 3.2 Obtain an S->D mutant of the phospho-site studied en 3.1 (possibly the GSK-3 site nearby S1397), and verify lack of binding to keratins and the effect on MTOC localization in vivo. 3.3 Assess depolarization of apical plasma membrane proteins, changes in vectorial membrane delivery and tight junction function after blocking the attachment of MTOCs to apical IPs. The long term goal of the project is to characterize a signaling pathway responsible for the depolarization of MTOCs and plasma membrane proteins in the late stages of the recovery after I/R and to design an intervention to revert it and improve recovery times after I/R. Lay statement: Acute renal failure affects 5% of patients admitted in hospitals and is a leading cause for the need of kidney transplants. In this project we analyze the structure and signaling that organizes the cytoskeleton during the second half of the recovery and ways to possibly shorten the period of kidney failure and to improve its recovery. The concepts can also be applied to the recovery of kidney transplants, that also suffer the consequences of temporal lack of blood supply.