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.

项目总结