My long-term career objectives are to apply fundamental concepts of cell and molecular biology to understand the role of cholangiocytes, the epithelial cells lining intrahepatic bile ducts, in health and disease. The current proposal focuses on the interactions between cholangiocytes and Cryptosporidium parvum, a coccidian parasite in the phylum Apicomplexathat has been associatedwith infection of the gastrointestinal, biliary, and respiratory epithelium. The primary goal of this proposal is to understand the mechanism and physiological consequences of localized, pathogen-induced water influx at the apical membrane of cholangiocytes, a process that may have broad implications in plasma membrane remodeling. Our preliminary observations support the central hypothesis that C. parvum cellular invasion requires efficient host cell membrane remodeling involving Myosin ll-dependent, exocytic insertion of vesicles containing Aquaporin 1 (Aqp1) and Na+/Glucose Cotransporter (Sgltl). We will employ complementary biochemical, molecular, and morphological approaches to address three Specific Aims to test the hypotheses that: (i) Myosin Haand lib are involved in the docking and fusion of vesicles containing Aqpt and Sgltl at infection sites on the apical cholangiocyte membrane via the direct interactions between Myosin II isoforms and vesicles containing Sgltl and Aqp1;(ii) The Cdc42 effector Myotonic Dystrophy Kinase-Related Cdc42- binding kinase (MRCK) and Myosin Light Chain Kinase (MLCK) contribute to the localized phosphorylation of Myosin Light Chain at C. parvum invasion sites;and, (iii) Myosin ll-mediated exocytic insertion of Aqp1 and Sgltl containing vesicles into the cholangiocyte apical membrane is involved in C. parvum induced localized water influx, a process facilitating localized membrane protrusion via promoting efficient actin dynamics at the site of infection. Thus, the proposed experiments will define the molecular mechanism by which the host actomyosin cytoskeleton facilitates exocytic insertion of Aqp1 and Sgltl at the infection sites on the apical membrane of cholangiocytes, and how the resultant water influx contributes to efficient membrane protrusion required for C. parvum cellular invasion, a phenomenon relevant to the molecular mechanisms of apical membrane modifications in general.
