Myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The intestinal epithelium creates a protective barrier separating the luminal content and the immune system in the gut. Leakiness of the intestinal epithelial barrier and inefficient healing of mucosal wounds represent common manifestations of inflammatory bowel diseases (IBD). Integrity of the gut barrier is mediated by different adhesive structures, including adherens junctions (AJ), tight junctions (TJ) and focal adhesion (FA). Functions of these adhesive structures depend on their association with the cortical actin cytoskeleton and particularly, to a major actin motor protein, non-muscle myosin II (NM-II). While biochemical mechanisms that regulate activity of NM-II in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. In this study, we focused on UNC-45A, a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis in parallel to diminished expression of NM-IIA and NM-IIC isoforms. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired AJ and TJ assembly, and cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC barrier function, junctional assembly and migration. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.