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.
Autosomal Dominant Polycystic Kidney Disease (ADPKD), in which the kidneys develop large cysts that may rupture under traumatic strain, is the most common inherited kidney disease and a leading cause of kidney failure. This project aims to determine how a disrupted signaling pathway in ADPKD may contribute to loss of structural integrity in kidney by altering the way that kidney cell-cell junctions respond to strain. The results of this project will inform our understanding of how mutations in ADPKD may alter cell-cell adhesion, and more broadly how cells maintain adhesion with their neighbors in the face of external strain.
