Proposed studies will continue the investigation of the organization and function of the cytoskeletal apparatus associated with the brush border (BB) membrane of intestinal epithelial cells. Studies will include the characterization of CA++ dependent phosphorylation of BB-myosin and its role in brush border motility and function in vitro and in vivo. Experiments will include characterization of phosphorylated BB-myosin and isolation and characterization of BB-myosin light chain kinase. Conditions for activation of BB-myosin kinase in isolated brush borders and intact epithelial cells will be determined, and the motility and changes in ultrastructure which accompany activation of BB-actomyosin will be analyzed. We will continue the characterization of microvillus (MV) core structure and proteins which include calmodulin, and subunits of 105k (the calmodulin binding protein), 95k (CA++ dependent actin binding protein responsible for core solation) and 68k-daltons. We will also continue studies on BB tropomyosin, a protein of the terminal web region of the BB. The effect of purified MV core proteins on actin assembly will be analyzed by a quantitative electron and light microscopy assay involving nucleated assembly from the two ends of the acrosomal filament bundle of Limulus sperm. We will also analyze the synthesis of actin and other contractile proteins in vivo in isolated epithelial cells and intact intestinal tissue. These studies will include localization of the sites of incorporation of newly synthesized actin into MV core filaments by cell fractionation techniques and EM autoradiography. Finally we will conduct a comparison of renal and intestinal brush borders from avian and mammalian tissues with respect to structure, contractile proteins, and CA++ dependent regulation of structure and contractility. These studies will provide information not accessible in other nonmuscle systems regarding the regulation of cytoskeletal structure and contractility. Results from the proposed studies will also provide important information regarding the role of the BB contractile apparatus in nutrient and electrolyte absorption normal and rachitic animals.
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