The actin-based cytoskeleton plays essential roles in a wide range of cellular phenomena including generation of cell shape, polarity , motility, cell signal transduction mechanisms, transport and localization of organelles and mRNAs, and modulation of the mechanical and physiological properties of cell membranes. The actin-based cytoskeletal apparatus that underlies the apical, microvillous or brush border (BB) of the intestinal epithelial cell is among the most high ordered cytoskeletal arrays in nature. The BB cytoskeleton has served for decades as a model system to investigate the organization, composition and functional properties of the actin-based cytoskeleton. The proposed studies will focus on several actin-based molecular motors (myosins) that are major components of the BB cytoskeleton in both vertebrates and Drosophila. The myosins to be investigated are class I myosins, one of fifteen known structurally microvillous by bridges composed of BB myosin-I (BBMI), which contains several bound calmodulin light chains, is among the best biochemically characterized myosins, yet nothing is known regarding its functions in vivo. Proposed functions include a role for this myosin in maintaining apical membrane polarity through vesicle transport or facilitation of nutrient absorption either by generation of microvillar movements or through mechanochemical regulation of membrane transporters in the BB membrane. These and other hypotheses will be tested through the phenotypic characterization of mice lacking the gene for BBMI (mice were made by collaborators). Studies will include phenotypic characterization BBMI-/- mice with respect to eneterocyte morphology, polarity, cytoskeletal composition and stability. Effects on intestinal functions including nutrient transport and response to intestinal pathogens and mucosal injury will be examined. In vivo dynamics of BBMI will be determined through visualization of GFP-tagged BMIO in the intestinal epithelial cell line, Caco2BBe. In Drosophila, the intestinal BB contains two class-I myosins, myosin-Ib (MIb) and myosin-Ia (MIa). Preliminary analysis suggests that both of these myosins are essential. Animals lacking MIb, which like BBMI tethers the BB membrane to underlying cytoskeleton, die as growth arrested first instar larvae, a phenocopy of starvation. This suggests a critical role for MIb in nutrient absorption. Characterization of MIb and MIa will include generation of multiple mutant alleles and phenotypic assessment of effects on epithelial cell organization, polarity, BB structure/composition and assessment of effects on gut physiology.
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