The Role of Mechanosensitive Calcium Signaling in Remodeling of Epithelial Cell-Cell Junctions

Abstract

Epithelial cells line most of our body cavities, separating different compartments in our bodies, creating selective barriers, and protecting us from external pathogens. In epithelial tissues, cells experience a range of mechanical forces like fluid flow, tissue distention, and tissue compression or contraction, which occur both during development and normal organ function. All of these events require cells to sense the mechanical force and remodel their cell-cell junctions accordingly in order to maintain barrier function and structural integrity of the tissue. However, the precise mechanism by which epithelial barrier function is regulated in response to mechanical stimuli is unknown.

In the vertebrate epithelium, the most apically localized cell-cell junctions, called tight junctions (TJs), regulate barrier function by selectively allowing solutes and ions to pass through the space between cells, while the more basally localized adherens junctions (AJs) mechanically couple the cells together to maintain the structural integrity of the tissue. Using gastrula-stage Xenopus laevis embryos as a model for the vertebrate epithelium, previous work from our lab identified local, short-lived leaks in the epithelial TJ barrier, which occur in response to elongating cell-cell junctions. These leaks were rapidly repaired by reinforcement of TJ proteins, mediated by localized, transient activation of the small GTPase RhoA – termed “Rho flares”. But the mechanism by which cells sense the leak in the barrier and activate Rho flares locally at the site of the barrier leak remained unclear.

In this dissertation, I investigate how a mechanical cue is converted into biochemical signals to regulate barrier function in the vertebrate epithelium. Here, I found that a local calcium increase occurs at the site of barrier leaks in response to loss of the TJ protein ZO-1. Using intracellular calcium chelation and a mechanosensitive calcium channel (MSC) blocker, I show that a local calcium increase is required for robust activation of Rho flares to efficiently reinforce TJs at sites of local barrier breaches. Further, I show that MSC-dependent calcium influx is required to maintain global barrier function by regulating efficient repair of local barrier leaks through robust contraction of junctions. Thus, we propose that MSC-mediated calcium influx is a mechanism by which epithelial cells detect local leaks and regulate barrier function by activating Rho flares. Additionally, I explored the localization and function of a eukaryotic MSC, Piezo1, at cell-cell junctions. I found that Piezo1 localizes to AJs and regulates TJ barrier function during cell-generated tensile stress, possibly by strengthening cell-cell adhesion. Together, my work reveals MSC-mediated calcium signaling as part of a mechanotransduction pathway working at apical cell-cell junctions to regulate barrier function. In addition to revealing a novel role for intracellular calcium signaling in regulating TJ barrier function in a mature epithelium, application of my research could help identify new targets to treat chronic inflammation associated with impaired cell-cell junctions in response to aberrant mechanotransduction.