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。它们控制着水、电解质和营养物质 动态平衡，所有这些在慢性肾脏疾病中都是错乱的。这些特化的上皮细胞需要高度的 决定细胞形态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，肌动蛋白 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。小分子Rho GTPase rac1是一种多功能的分子开关，也是Rho GTP酶的主调节子 肌动蛋白细胞骨架。我们最近证明了rac1是维持肌动蛋白细胞骨架完整性所必需的， 成熟的集合管(CD)上皮的上皮极性和细胞形状。目前尚不清楚肌动蛋白是如何 细胞骨架在上皮细胞修复中受到调控，以及rac1在这一过程中扮演什么角色。用于协调 上皮管再生，上皮细胞需要在细胞周期中快速进行，并在 通过进行定向有丝分裂来纠正沿组织平面的方向。肾上皮修复的一个关键步骤是 激活主有丝分裂激酶Cyclin B-CDK1复合体，推动G2/M转变并准备 有丝分裂的肌动蛋白细胞骨架。在密闭的上皮空间中正确地进行有丝分裂是一项具有挑战性的工作 过程，这需要细胞聚集起来反对它们的邻居。这种所谓的有丝分裂圆化取决于 在肌动蛋白细胞骨架上形成致密的收缩肌球蛋白皮质，该皮质被拴在细胞膜上 通过ERM蛋白(Ezrin)，一个已知的rac1及其主要效应蛋白p21激活的激酶(Pak1)的靶标。中的缺陷 有丝分裂形态导致细胞周期延迟、机械缺陷、细胞死亡和异常修复。显示肌动蛋白 细胞骨架动力学是分子协调的，细胞形态通过什么方式与细胞周期有关 在肾脏修复的这些关键生物学过程中的控制尚不清楚。我们删除了集合中的rac1 导管(AQP2Cre)或近端小管(γGTCre)和可逆性单侧输尿管梗阻(R-UUO) 或缺血再灌注性急性肾损伤(AKI-IRI)。功能和组织学评估显示 在这两款车型中，都需要使用rac1进行维修。光学清理和3D高分辨率成像显示， 突变的上皮细胞在损伤后不能恢复正常的形态和肌动蛋白组织。Repair in rac1 突变体表现出肌动蛋白依赖的有丝分裂形态的缺陷，有丝分裂图形错位，以及不能 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 Rac1是有丝分裂和Ezrin相关的有丝分裂的正常机械进展所必需的 上皮细胞。这形成了我们的假设的基础，即rac1控制着肌动蛋白依赖的有丝分裂圆化 通过其效应器pak1和连接蛋白Ezrin介导，促进细胞周期 组织修复，这将在以下目标中进行测试。目标1：确定rac1的机制 在体内调节肾上皮修复。目标2：确定rac1控制肾脏的机制 体外培养上皮细胞有丝分裂形态。