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.

偏振上皮管对于完整的肾脏至关重要。他们控制水，电解质和养分 稳态，所有这些都在慢性肾脏疾病中丧命。这些专业上皮需要高度 有组织的肌动蛋白细胞骨架决定细胞形状和功能。在肾上皮修复期间，肌动蛋白 增殖细胞的细胞骨架迅速重新组织，形成了复杂的极化结构 上皮管。小的Rho GTPase Rac1是一种多功能分子开关，是一个主调节器 肌动蛋白细胞骨架。我们最近证明了Rac1需要维持肌动蛋白细胞骨架完整性， 上皮极性和成熟收集管（CD）上皮的细胞形状。仍然未知肌动蛋白 细胞骨架在上皮细胞修复中受到调节，Rac1在此过程中起什么作用。进行协调 上皮管再生，上皮细胞需要在细胞周期中快速发展，并在 通过经历定向有丝分裂沿组织平面纠正方向。肾上皮修复的关键步骤是 主有丝分裂激酶Cyclin B – CDK1复合物的激活，该复合物驱动G2/M过渡并准备 肌动蛋白细胞骨架用于有丝分裂。在密闭的紧上皮空间中正确进行有丝分裂是一个挑战 过程，这需要细胞与邻居的围绕。这个所谓的有丝分裂圆形取决于 在肌动蛋白的细胞骨架上形成密集的收缩肌球蛋白皮质，该皮质被束缚在细胞膜上 通过ERM蛋白（Ezrin），Rac1的已知靶标及其主要效应子P21激活激酶（PAK1）。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。肌动蛋白 细胞骨架动力学是分子协调的，通过意味着细胞形状与细胞周期相关 在肾脏修复中这些关键的生物过程中的控制尚不清楚。我们在收藏中删除了Rac1 导管（AQP2CRE）或近端管（γGTCRE），并进行了可逆的单侧输尿管异常（R-UUOO） 或缺血再灌注诱发急性肾脏损伤（AKI-IRI）。功能和组织学评估表明 在这两种型号中，Rac1都是维修所必需的。光学清除和3D高分辨率成像表明 突变上皮无法恢复受伤后正常形态和肌动蛋白组织。 Rac1维修 突变体显示了肌动蛋白依赖性有丝分裂形态的缺陷，错位的有丝分裂数字以及无法 由于G2/M细胞周期缺陷而增殖。 3D高分辨率在体外对细胞分裂的实时成像表明， RAC1是有丝分裂的正常机械进展和与肾脏相关的有丝分裂舍入的 上皮细胞。这种形式是我们假设Rac1控制肌动蛋白依赖性有丝分裂舍入的基础 通过其效应子Pak1和接头蛋白EZRIN进行介导，以促进细胞循环。 组织修复，将在以下目的中进行测试。目标1：定义Rac1的机制 调节体内肾上皮修复。目标2：定义Rac1控制肾脏的机制 体外上皮有丝分裂形态。