f, g Recoil measurements (f) and initial recoil velocities (g) at junctions in control and SRGAP1 KD cells after laser ablation. SRGAP1. We further demonstrate that this mechanism is co-opted by hepatocyte growth factor to promote junctional relaxation and motility in epithelial collectives. Together, our findings identify a novel function of cortactin as a regulator of RhoA signaling that can be utilized by morphogenetic regulators for the active downregulation of junctional contractility. Introduction Epithelial adherens junctions are contractile structures, where coupling of actomyosin to E-cadherin generates junctional tension that promote cell?cell adhesion and assembly of the specialized adherens junction of the zonula adherens (ZA)1, 2. In addition, the coupling of contractility to adhesion participates in a variety of morphogenetic processes, such as apical constriction and epithelial furrowing3, 4. The functional consequences of applying contractile force at junctions have commonly Ganirelix been studied when those forces are increased in some regulated fashion, or when coupling of contractility to adhesion is developmentally activated3. However, other developmental HDAC6 circumstances entail the downregulation of cell?cell junctions. In the extreme case, cell?cell contacts may break down altogether when E-cadherin expression is suppressed during epithelial-to-mesenchymal transitions5. However, there are many other instances where cells rearrange while maintaining E-cadherin-based contacts with one another4. For example, when border cell clusters migrate in the egg chamber6, E-cadherin contacts persist between border cells and the nurse cells that they move through and are, indeed, necessary for invasive movement to occur7. Similarly, functional downregulation of Ganirelix adherens junctions is thought to underlie the morphogenetic changes seen when cultured mammalian epithelial cells are stimulated with Hepatocyte Growth Factor (HGF)8, 9, which plays a vital role in organ development and wound repair10, 11. However, whether junctional contractility might also be modulated in these circumstances remains an open question. In cultured epithelial cells, biogenesis of the junctional actomyosin cytoskeleton is necessary for the generation of contractility. This involves diverse processes that must be coordinated at the junctional cortex, including actin assembly12, 13, filament Ganirelix network reorganization14, and activation of non-muscle myosin II (NMII) by junctional RhoA15. Cortactin is a scaffolding protein that bears multiple potential protein?protein interaction domains and can influence many steps in cytoskeletal biogenesis16. It associates with the E-cadherin molecular complex and concentrates at sites of junctional contractility, notably when epithelia assemble a ZA, where it promotes actin assembly17, 18. Thus, cortactin presents as an attractive candidate to regulate actomyosin at the junctional cortex. Cortactin is a tyrosine and serine phosphoprotein. Originally identified as a substrate for Src family kinases (SFK), cortactin is targeted by a number of protein kinases and Ganirelix phosphatases that function in different cellular processes16. Tyrosine phosphorylated cortactin is readily detected at cell?cell junctions, potentially generated by SFK activity in this location19. Indeed, expression of phosphomimetic mutants suggested that tyrosine phosphorylated cortactin might support junctional integrity downstream of junctional Src signaling20, 21. But how the tyrosine phosphorylated status of cortactin influences junctional biology remains poorly characterized. Here, we have identified a novel role for the tyrosine-dephosphorylated form of cortactin as a negative regulator of junctional contractility. We report that tyrosine-dephosphorylated cortactin downregulates junctional RhoA signaling by promoting the junctional accumulation of SRGAP1, a RhoA antagonist. We further show that this pathway is utilized by HGF to relax junctions and promote epithelial locomotility. Results Tyrosine non-phosphorylated cortactin downregulates ZA tension To begin, we tested how depleting cortactin affected junctional contractility in Caco-2 cells. Lentiviral shRNA reduced cellular cortactin (Supplementary Fig.?1a) and junctional cortactin staining detectable by immunofluorescence (IF; Supplementary Figs.?1d, e and 2) by ~?90%. We then used laser ablation to cut junctions marked by E-cad-GFP (expressed on an E-cad shRNA background; Fig.?1a) and measured the instantaneous velocity of recoil as an index of tension (Fig.?1b)15. As previously reported17, 18, cortactin knockdown (KD) decreased E-cadherin concentration at the apical ZA (Fig.?1c, d) without altering overall cellular or surface levels of the protein (Supplementary Fig.?1a, b). Fluorescence recovery after photobleaching (FRAP) revealed that the immobile fraction of E-cad-GFP (tagged at the endogenous locus by CRISPR-based genome editing; see?Supplementary Methods) was also reduced by cortactin KD (Fig.?1g, h), suggesting that.