Mechanosensitive calcium flashes promote sustained RhoA activation during tight junction remodeling

Abstract

Epithelial cell–cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed “Rho flares,” but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.

p115RhoGEF activates RhoA to support tight junction maintenance and remodeling

“…Rho flares in p115.S KD embryos displayed reduced intensity and duration of active RhoA at sites of TJ protein loss ( Figure 2 , A-C’, and Video 2 ). This reduction in active RhoA at flares led us to test whether downstream TJ repair is also compromised (Stephenson et al ., 2019; Varadarajan et al ., 2022). Indeed, the rapid junction contraction that immediately follows Rho flares in control embryos was absent in p115.S KD embryos ( Figure 2 , D-D’ ).…”

“…Rho flares rapidly repair the TJ barrier through localized actomyosin accumulation, which promotes junction contraction and TJ protein reinforcement (Stephenson et al ., 2019). Recent work revealed that intracellular calcium flashes, along with the mechanical stimulus of junction elongation, are early events in the Rho flare signaling pathway (Varadarajan et al ., 2022). These calcium flashes, mediated through mechanosensitive calcium channels, are required for sustained RhoA activation to support efficient repair and reinforcement of the TJ barrier (Varadarajan et al ., 2022).…”

“…Recent work revealed that intracellular calcium flashes, along with the mechanical stimulus of junction elongation, are early events in the Rho flare signaling pathway (Varadarajan et al ., 2022). These calcium flashes, mediated through mechanosensitive calcium channels, are required for sustained RhoA activation to support efficient repair and reinforcement of the TJ barrier (Varadarajan et al ., 2022). However, a crucial step of the Rho flare TJ remodeling pathway remains unclear: what activates RhoA flares?…”

Compressive stress drives adhesion-dependent unjamming transitions in breast cancer cell migration

“…One open question is how compressive stress leads to increased recruitment of E-cadherin to promote cell-cell adhesion. Mechanical stimuli are known to remodel epithelial cell-cell junctions by junction elongation and contraction mediated via mechanosensitive channels (Varadarajan et al ., 2022). It is also well documented that adherens junctions become reinforced when cells are under tension (Borghi et al ., 2012; Charras and Yap, 2018; Pinheiro and Bellaíche, 2018).…”

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells

“…At sites of TJ breaches markers like ZO-1 disappear, and they reappear shortly after the recruitment of active RhoA ( Stephenson et al, 2019 ). This repair process is preceded by a local increase in the intracellular Ca 2+ concentration, suggesting that mechanosensitive Ca 2+ channels act as sensors of TJ breaches, and that a local increase in intracellular Ca 2+ activates RhoA at the TJs ( Varadarajan et al, 2022 ) ( Figure 5 ). These findings thus provide strong evidence that in reponse to local perturbations of TJ integrity, RhoA is recruited and/or activated locally to induce contractility of the actin cytoskeleton to support the repair of TJs and to re-establish the epithelial barrier function.…”