Members of the myosin family of actin filament based molecular motors perform a wide range of critical cellularfunctions including cellular and organelle motility, tension generation, signal transduction and transcriptionalregulation. This project will explore in vivo functions of myosins in the intestinal epithelial cell (enterocyte) withparticular focus on Myo9b, a myosin implicated in inflammatory bowel disease (IBD), and on the functionalsynergy between Myo6, the sole minus-end motor linked to the IEC brush border membrane (BBM), andMyo1a, the principal BBM-associated plus-end motor. These studies will utilize available mouse mutants foreach of these myosins, as well as a Myo1a/Myo6 double mutant (DM). Myo9b contains a GTPase activatingprotein (GAP) domain for Rho in its tail. It is likely that Myo9b-linked IBD is due to hyperactivation of Rho, acritical regulator of the actin cytoskeleton. In vitro studies have implicated Myo9b in enterocyte monolayerwound healing and maintaining tight junction (TJ) integrity and paracellular barrier properties. Preliminarystudies indicate that Myo9b knock out (KO) mice under unstressed conditions exhibit hallmark symptoms ofIBD including presence of erythrocytes in the gut lumen, greatly elevated enterocyte cell death and apoptosis,inter-enterocyte invasion of immune response cells and altered junctional properties. Taken together theseobservations indicate that the Myo9b KO should be a powerful and novel animal model for IBD, and becausethese symptoms occur in both small and large intestine, for Crohn's disease in particular. Proposed studieswill provide a thorough phenotypic characterization of this IBD model, including effects on enterocyte polarity,cytoskeletal organization, and of particular importance to IBD, TJ composition/organization. The mucosalbarrier properties of the Myo9b KO will be quantified and the predicted hyper-sensitive response to mucosalinflammation and injury will be assessed. Studies to investigate the functional synergy between Myo1a andMyo6 will include pilot studies to test the hypothesis that Myo1a suppresses the tumor promoting activity ofMyo6 through sequestration of this putative proto-oncogene to the BB. Other studies will quantify the relativecontributions of Myo6 versus Myo1a to the tethering of the BBM to the actin cytoskeleton, and to the in vivogeneration and shedding of BBM luminal vesicles that may be critical for protection from bacterial pathogens.Another set of studies will investigate the molecular bases for potential restoration of WT BBM function (e.g.Ca2+ transport, lipid raft organization, BBM micro-viscosity, CFTR Cl- channel activity) when both Myo1a andMyo6 are absent. Finally the synergistic roles of Myo1a and Myo6 endocytic and exocytic trafficking of BBMproteins (e.g. cystic fibrosis transmembrane conductance regulator, Na+/phosphate exchanger 2b,Toll-like receptor4) will be explored.
The project focuses on defining the in vivo functions for myosins of the intestinal epithelial cell (IEC). Studies include establishment of a new animal model for inflammatory bowel disease (IBD) through characterization of mice lacking a myosin linked to IBD. Other studies focus on defining the molecular basis for the synergistic involvement of two major myosins of the IEC in suppressing intestinal tumors. The normal synergistic functions of these two myosins in IEC physiology, membrane traffic and cytoskeleton-membrane interaction will also be investigated.
