PROJECT SUMMARY: Disruption of the intestinal epithelial barrier in patients with inflammatory bowel diseases (IBD) involves disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ), along with formation of mucosal wounds due to excessive epithelial cell death. Restoration of the gut barrier (epithelial restitution) is critical for achieving remission of the disease and it involves mucosal wound healing and AJ/TJ reassembly. Both processes are mediated by common molecular events, most notably, dramatic remodeling of the epithelial actomyosin cytoskeleton. An unconventional myosin 18A (Myo18A) is a unique PDZ-domain containing scaffolding protein that acts as an essential regulator of actin filaments and their motor, non-muscle myosin II (NM II). Myo18A is known to control neuronal and muscle morphogenesis, as well as tumor growth and dissemination, however its functions in the gut have not been previously investigated. Our preliminary data suggests that Myo18A is abundantly expressed in normal human intestinal epithelium and it is a novel component of epithelial junctions, Furthermore, Myo18A depletion leads to various functional defects, including disruption of the epithelial barrier, attenuation of wound healing and impairment of 3-D epithelial morphogenesis. Importantly, we observed a marked downregulation of Myo18A expression in the intestinal epithelium of ulcerative colitis patients. This exciting preliminary data provides a strong scientific premise for the following innovative hypothesis: Myo18A is a novel regulator of the intestinal epithelial barrier integrity and restitution, and its depletion promotes gut leakiness and inhibits mucosal healing in IBD patients. This hypothesis will be tested in the following Aims: (1) to determine the roles of Myo18A in the regulation of intestinal epithelial cell migration and ECM adhesion; (2) to delineate the mechanisms of Myo18A-dependent assembly of the epithelial barrier and establishment of the apico-basal cell polarity; 3) to examine the roles of Myo18A in regulating disruption and restitution of the intestinal epithelial barrier in vivo. The Aims will be accomplished by studies utilizing in vitro intestinal epithelial cell monolayers, ex vivo colonic organoids generated from IBD mucosa and in vivo murine models of epithelial injury and restitution. Roles of Myo18A will be examined by a combination of functional (permeability measurements, wound healing), biochemical (immunoblotting, detergent fractionation), imaging (FRET biosensors, super-resolution microscopy), and genetic (CRISPR/Cas9 gene editing, overexpression of deletion mutants, knockout mice) approaches. Significance: the proposed study will yield novel insights into the mechanisms that regulate intestinal epithelial injury and restitution during inflammation. It will also identify new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases.