Rac1 and the actin cytoskeleton in renal tubular repair

Rac1 和肌动蛋白细胞骨架在肾小管修复中的作用

• 批准号: 10739610
• 负责人: Fabian Maximilian Bock
• 金额: $ 16万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739610
• 关键词: 3-Dimensional; Actins; Actomyosin; Acute Renal Failure with Renal Papillary Necrosis; Architecture; Biological Process; CDC2 gene; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cell Shape; Cell division; Cell membrane; Cells; Cellular Morphology; Chronic Kidney Failure; Complex; Cyclin B; Cytoskeleton; Data; Defect; Development; Duct (organ) structure; Ductal Epithelium; ERM protein; Electrolytes; Epithelial Cells; Epithelium; G2/M Transition; Histologic; Homeostasis; In Vitro; Injury; Ischemia; Kidney; Kidney Diseases; Knowledge; Lead; M cell; Mechanics; Mediating; Mitosis; Mitotic; Modeling; Molecular; Morphology; Natural regeneration; Nutrient; Obstruction; Optics; Phosphotransferases; Play; Process; Proliferating; Proteins; Reperfusion Therapy; Resolution; Role; Shapes; Testing; Tissues; Tube; Tubular formation; Water; Work; epithelial repair; epithelium regeneration; high resolution imaging; in vivo; kidney repair; live cell imaging; molecular dynamics; mutant; new therapeutic target; novel; p21 activated kinase; renal epithelium; repaired; rho GTP-Binding Proteins; tissue repair

Polarized epithelial tubes are critical for an intact kidney. They control water, electrolyte, and nutrient homeostasis, all of which are deranged in chronic kidney disease. These specialized epithelia require a highly organized actin cytoskeleton that determines cellular shape and function. During renal epithelial repair, the actin cytoskeleton of proliferating cells is rapidly re-organized to form the complex polarized architecture of an epithelial tube. The small Rho GTPase Rac1 is a multifunctional molecular switch and a master regulator of the actin cytoskeleton. We recently demonstrated that Rac1 is required to maintain actin cytoskeletal integrity, epithelial polarity, and cell shape of the mature collecting duct (CD) epithelium. It is still unknown how the actin cytoskeleton is regulated in epithelial cell repair and what role Rac1 plays in this process. For coordinated epithelial tube regeneration, epithelial cells need to rapidly progress through the cell cycle and divide in the correct direction along the tissue plane by undergoing oriented mitosis. A critical step in renal epithelial repair is the activation of the master mitotic kinase cyclin B–CDK1 complex which drives the G2/M transition and prepares the actin cytoskeleton for mitosis. Undergoing mitosis correctly in a confined tight epithelial space is a challenging process, which requires the cells to round up against their neighbors. This so-called mitotic rounding depends on the actin cytoskeleton forming a dense contractile actomyosin cortex, which is tethered to the cell membrane by ERM Proteins (Ezrin), a known target of Rac1 and its main effector p21-activated kinase (Pak1). Defects in mitotic morphology lead to cell cycle delays, mechanical defects, and cell death and abnormal repair. How actin cytoskeletal dynamics are molecularly coordinated and by what means cell shape is connected to cell cycle control during these critical biological processes in kidney repair is not known. We deleted Rac1 in the collecting duct (AQP2Cre) or proximal tubule (γGTCre) and performed reversible unilateral ureteric obstruction (R-UUO) or ischemia-reperfusion induced acute kidney injury (AKI-IRI). Functional and histological assessment indicated that Rac1 is required for repair in both models. Optical clearing and 3D high-resolution imaging revealed that the mutant epithelium was unable to restore normal morphology and actin organization post-injury. Repair in Rac1 mutants showed defects in actin-dependent mitotic morphology, misplaced mitotic figures, and the inability to proliferate due to a G2/M cell cycle defect. 3D high-resolution live imaging of cell division in vitro revealed that Rac1 is required for normal mechanical progression of mitosis and Ezrin-associated mitotic rounding in renal epithelial cells. This forms the basis for our hypothesis that Rac1 controlled actin-dependent mitotic rounding mediated via its effector Pak1 and the linker protein Ezrin is required to promote cell cycling during tissue repair, which will be tested in the following aims. Aim 1: Define the mechanisms whereby Rac1 regulates renal epithelial repair in vivo. Aim 2: Define the mechanism whereby Rac1 controls renal epithelial mitotic morphology in vitro.

极化的上皮管对完整的肾脏至关重要。它们控制水、电解质和营养 体内平衡，所有这些都在慢性肾病中紊乱。这些特化的上皮细胞需要高度的 决定细胞形状和功能的有组织的肌动蛋白细胞骨架。在肾上皮修复过程中， 增殖细胞的细胞骨架迅速重组，形成复杂的极化结构， 上皮管小的Rho GTCRac Rac 1是一种多功能分子开关，也是一种主要的调节剂。 肌动蛋白细胞骨架我们最近证明Rac 1是维持肌动蛋白细胞骨架完整性所必需的， 上皮极性和成熟集合管（CD）上皮的细胞形状。目前还不清楚， 细胞骨架在上皮细胞修复中受到调节，Rac 1在这一过程中发挥了什么作用。协调 上皮管再生，上皮细胞需要通过细胞周期快速进展，并在细胞周期中分裂。 通过进行定向有丝分裂来沿着组织平面沿着正确方向。肾上皮修复的关键步骤是 主有丝分裂激酶细胞周期蛋白B-CDK 1复合物的激活，该复合物驱动G2/M转换并准备 有丝分裂的肌动蛋白细胞骨架。在狭窄的上皮间隙中正确进行有丝分裂是一个挑战， 这一过程要求细胞与它们的邻居进行围捕。这种所谓的有丝分裂圆形化取决于 在肌动蛋白细胞骨架上形成一个紧密的收缩性肌动球蛋白皮质，它被拴在细胞膜上 ERM Proteins（Ezrin），Rac 1及其主要效应子p21激活激酶（Pak 1）的已知靶点。缺陷 有丝分裂形态导致细胞周期延迟、机械缺陷以及细胞死亡和异常修复。如何行动 细胞骨架动力学是分子协调的，细胞形状是通过什么方式与细胞周期相联系的？ 在肾修复的这些关键生物过程中的控制是未知的。我们在收集中删除了Rac 1 输尿管近端小管（γGTCre）或近端小管（AQP 2Cre），行可逆性单侧输尿管梗阻（R-UUO） 或缺血-再灌注诱导的急性肾损伤（AKI-IRI）。功能和组织学评估表明 两种型号都需要Rac 1进行维修。光学透明和3D高分辨率成像显示， 突变上皮不能恢复损伤后的正常形态和肌动蛋白组织。Rac 1中的修复 突变体在肌动蛋白依赖的有丝分裂形态上表现出缺陷，有丝分裂图错位， 由于G2/M细胞周期缺陷而增殖。体外细胞分裂的3D高分辨率实时成像显示， Rac 1是肾细胞有丝分裂和Ezrin相关有丝分裂变圆的正常机械进展所必需的。 上皮细胞这构成了我们假设Rac 1控制肌动蛋白依赖的有丝分裂圆化的基础 通过其效应蛋白Pak 1和连接蛋白Ezrin介导的细胞周期调节是必需的，以促进细胞周期， 组织修复，将在以下目标中进行测试。目标1：确定Rac 1 调节体内肾上皮修复。目的2：确定Rac 1控制肾功能的机制 体外上皮细胞有丝分裂形态。