Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling

To preserve barrier function, cell-cell junctions must dynamically remodel during cell shape changes. We have previously described a rapid tight junction repair pathway characterized by local, transient activation of RhoA, termed 'Rho flares,' which repair leaks in tight junctions via promoting local actomyosin-mediated junction remodeling. In this pathway, junction elongation is a mechanical trigger that initiates RhoA activation through an influx of intracellular calcium and recruitment of p115RhoGEF. However, mechanisms that tune the level of RhoA activation and Myosin II contractility during the process remain uncharacterized. Here, we show that the scaffolding protein Anillin localizes to Rho flares and regulates RhoA activity and actomyosin contraction at flares. Knocking down Anillin results in Rho flares with increased intensity but shorter duration. These changes in active RhoA dynamics weaken downstream F-actin and Myosin II accumulation at the site of Rho flares, resulting in decreased junction contraction. Consequently, tight junction breaks are not reinforced following Rho flares. We show that Anillin-driven RhoA regulation is necessary for successfully repairing tight junction leaks and protecting junctions from repeated barrier damage. Together, these results uncover a novel regulatory role for Anillin during tight junction repair and barrier function maintenance.