Movement is a ubiquitous feature of all cells and includes such processes as mitosis, cytokinesis, locomotion of single cells, axonal transport, and membrane trafficking. Both the actin- and microtubule-based cytoskeleton and their sets of associated mechanochemical motors provide the means with which the shape, locomotion and form of cells is established We have investigated the intestinal enterocyte as a model polarized epithelial cell because of its stereotyped and well- characterized actin and microtubule cytoskeleton and its carefully analyzed pathways of Golgi membrane trafficking. Proper delivery of sorted membrane proteins is essential to the normal absorptive functions of the intestinal epithelium and several congenital human diseases of the small intestine, including sucrase-isomaltase deficiency and microvillar atrophy, are likely the result of improper delivery or sorting of membrane proteins. The actin-based motor, myosin I, forms cross-links between the actin filament bundle and the overlying membrane of the brush border microvilli. Myosin I may be a motor, in the highly cross-linked cell cortex, for the movement of membrane vesicles destined for delivery- to the plasma membrane. Although the Golgi apparatus requires intact microtubules and associated motors dynein and kinesin to maintain Golgi structure and proper direct and indirect delivery of membranes, microtubules do not extend to the actin-rich cortex. Therefore, proper membrane delivery might require the coupling of the microtubule and actin based motor systems. We have shown that enterocyte Golgi stacks and other cytoplasmic membranes possess myosin I, that trans-Golgi membranes possess myosin I and dynein, and that Golgi membranes bind to actin filaments and microtubules in an ATP-sensitive manner. Preliminary evidence shows that enterocyte Golgi membranes move in vitro along microtubules and that membrane-associated myosin I can be phosphorylated, which in other systems effects its activities. We propose a series of studies to analyze the potential role of phosphorylation on molecular motor activity and association with membranes. Both in vivo and in vitro phosphorylation sites and stoichiometry will be mapped. A comparable analysis of dynein phosphorylation and its effects on activity and membrane binding will be performed. Potential membrane proteins which bind myosin I or dynein will be investigated. In vitro motility and its regulation on both microtubules and actin filament bundles will be studied. The use of an in vitro Golgi budding assay using liver as well as enterocytes will be exploited in these studies. Other cell lines, where manipulation of Golgi regulatory factors are available, will be studied for regulation of cytoskeletal motor function. Finally, analysis of the in vitro and in vivo role of tropomyosins as regulatory proteins will be pursued.
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