AbstractThe intestinal epithelium is vital to maintain the barrier between the body and the outside world and membranetrafficking is essential for both the development and maintenance of this barrier. Abnormal membranetrafficking can result compromised barrier leading to intestinal disease including inflammatory bowel disease.Autophagy is a specialized membrane trafficking process that allows cells to respond to changes inmetabolism. In addition autophagy is important for maintenance of the epithelial barrier and the innate immuneresponse mediating the isolation and degradation of intracellular pathogens. Furthermore LC3-associatedphagocytosis (LAP) is important for uptake and degradation of pathogens and dysfunction of this pathway isassociated with hyperinflammation. Importantly variants of proteins in the autophagic and LAP pathways havebeen linked to increased susceptibility to Inflammatory Bowel Disease (IBD) and particularly Crohns diseasealthough the molecular mechanisms that underlie this connection remain incompletely understood. MAMDC4 isan integral membrane protein that localizes to endosomes of the intestinal epithelium. Deletion of MAMDC4compromises intestinal enterocyte morphology and RNAseq studies have indicated that MAMDC4 is down-regulated in IBD. In previous work we found that MAMDC4 interacts with the small GTPase Rab14. Inprofessional phagocytes Rab14 is recruited to phagosomes and prevents phagosome-lysosome fusion but itsrole in the intestinal epithelium is unknown. In our preliminary data we show that Rab 14 is present onautophagosomes and both Rab14 and MAMDC4 are present on membranes containing invading bacteria.Furthermore deletion of MAMDC4 results in accumulation of autophagy and lysosomal markers on tubularmembranes. These results suggest that MAMDC4 and Rab14 act in a molecular network to maintain mucosalimmunity through control of autophagy or LAP. In this proposal we will use intestinal epithelial cells in cultureorganoid culture and patient-derived organoids to define the role of autophagy and/or LAP in intestinalhomeostasis.