Molecular Basis of Renal Epithelial Cell-Cell Adhesion

肾上皮细胞-细胞粘附的分子基础

• 批准号: 10363722
• 负责人: Vipul Vachharajani
• 金额: $ 2.28万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10363722
• 关键词: Acute; Address; Adhesions; Adhesives; Affect; Architecture; Area; Autosomal Dominant Polycystic Kidney; Binding; Biological Assay; Biophysical Process; Cadherins; Caliber; Canis familiaris; Cell Adhesion; Cell Adhesion Molecules; Cell-Cell Adhesion; Cells; Clinical; Collection; Communication Research; Complex; Cyst; Cystic kidney; Cytoskeleton; Development; Disease; Dissociation; E-Cadherin; Ensure; Epithelial; Epithelial Cells; Equilibrium; Fluorescence; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Proteins; Goals; Individual; Inherited; Integral Membrane Protein; Intercellular Junctions; Kidney; Kidney Diseases; Kidney Failure; Lead; Lipid Bilayers; Magnetism; Measures; Mechanics; Mediating; Membrane; Microscopy; Molecular; Mutation; Physicians; Physiology; Population; Process; Protein Subunits; Proteins; Regulation; Research Training; Resolution; Rupture; Scientist; Signal Pathway; Signal Transduction; Sum; Supporting Cell; System; Testing; Time; Tissues; Training; Trauma; Tubular formation; Twin Multiple Birth; Weight-Bearing state; Work; base; biophysical techniques; biophysical tools; career; contrast imaging; experience; extracellular; kidney cell; kidney epithelial cell; mechanical load; polycystic kidney disease 1 protein; reconstitution; renal epithelium; resilience; response; sensor; single molecule; transmission process; urinary

Project Summary The goal of this project is to determine how renal tubular epithelial cells achieve robust cell-cell adhesion when faced with external forces. In the kidney, this occurs regularly as volume fluctuations distend the urinary collecting system to varying degrees. An extreme example occurs in autosomal-dominant polycystic kidney disease (ADPKD), the most common inherited renal disorder, where renal cysts can endure 1000-fold strain in diameter, but can rupture upon acute trauma, leading to other serious consequences. Approximately 85% of ADPKD cases are caused by mutations in the protein polycystin-1 (Pc-1), a putative atypical G-protein coupled receptor that is involved in intracellular signal transduction via sequestration of the G protein subunit G12. Relatively little is known about how dysregulated G12-mediated signaling in ADPKD leads to the physical compromise of cell-cell adhesion. This gap persists, in part, because even simple questions remain unanswered about how epithelial cells mechanically regulate cell-cell adhesions under strain. This proposal will address two such fundamental questions, using the case of ADPKD as a concrete example of how such regulation may be disrupted. To do so, I will make use of a semi-reconstituted system in which Madin- Darby Canine Kidney (MDCK) epithelial cells form junctions with supported lipid bilayers (SLBs) decorated with the cell adhesion molecule E-cadherin. This system enables both high resolution microscopy on live cells and precise application of externally applied forces using magnetic tweezers. Aim 1 will address the question of how cells ensure robust adhesion using the E-cadherin molecules that bind between cells. High resolution total internal reflectance fluorescence (TIRF) and reflectance interference contrast (RICM) imaging will be used to visualize the clustering of E-cadherin and the cell-SLB distance, respectively, as a function of applied force. Aim 2 will address the question of how cells transmit external loads through the collection of E-cadherin molecules. Fluorescent single-molecule tension sensors will be used to directly measure single-molecule force distributions as a function of externally applied load. Finally, Aim 3 will systematically perturb the Pc-1/G12 signaling axis to determine how cadherin-mediated adhesion and force transmission may be dysregulated in ADPKD. The results of this work will determine how signaling downstream of Pc-1 may contribute to the dysregulation of cell-cell adhesion in ADPKD, and, more broadly, reveal the biophysical mechanisms that epithelial cells use to maintain robust cell-cell adhesion even in the face of sometimes extreme external forces. When combined with a research training plan emphasizing development in research communication and incorporating continued clinical experience, this work will prepare me to pursue further training towards a career as an independent physician-scientist studying disrupted tissue architecture in disease.

项目摘要 该项目的目的是确定肾小管上皮细胞如何实现可靠的细胞粘附 面对外力时。在肾脏中，随着体积波动的扩大，这种情况会定期发生 在不同程度上收集系统。一个极端的例子发生在常染色体主导的多囊肾脏中 疾病（ADPKD）是最常见的遗传疾病，肾脏囊肿可以忍受1000倍的菌株 直径，但可能会在急性创伤时破裂，从而导致其他严重的后果。 大约85％的ADPKD病例是由蛋白质多囊蛋白1（PC-1）突变引起的 假定的非典型G蛋白偶联受体，该受体与细胞内信号转导通过隔离 G蛋白亚基G12。关于如何失调的G12介导的信号传导的知之甚少 ADPKD导致细胞粘附的物理妥协。这个差距仍然存在，部分原因是 关于上皮细胞如何机械调节菌株下的细胞 - 细胞粘合剂的问题仍然没有提示。 该提案将以ADPKD为具体示例，解决两个这样的基本问题 这种法规如何破坏。为此，我将利用一个半稳定的系统，其中madin- 达比犬肾（MDCK）上皮细胞与装饰有支撑的脂质双层（SLB）形成连接 细胞粘附分子电子钙粘着蛋白。该系统可以在活细胞和 使用磁性镊子精确应用外部施加力。 AIM 1将解决细胞如何使用E-钙粘蛋白分子确保鲁棒性的问题。 结合细胞之间。高分辨率总内反射率荧光（TIRF）和反射率干扰 对比度（RICM）成像将用于可视化E-钙粘蛋白和细胞-SLB距离的聚类， 分别是施加力的函数。 AIM 2将解决细胞如何传输外部负载的问题 通过E-钙粘蛋白分子的收集。荧光单分子张力传感器将用于 直接测量单分子力分布作为外部施加载荷的函数。最后，AIM 3将 系统地扰动PC-1/G12信号轴，以确定钙粘蛋白介导的粘合剂和力如何 ADPKD中的传输可能失调。 这项工作的结果将决定PC-1下游的信号如何有助于 ADPKD中细胞细胞粘附的失调，更广泛地揭示了生物物理机制 上皮细胞即使面对有时极端的外力，也用于维持健壮的细胞粘合剂。 当与研究培训计划结合在一起，强调研究沟通中的发展和 融合了持续的临床经验，这项工作将使我为进一步的职业做好准备 作为一个独立的身体科学家研究疾病中的组织结构的破坏。