MICROVILLAR CYTOSKELETAL-MEMBRANE INTERACTIONS

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The study of how the cytoskeleton interacts with the membrane will explain several cellular phenomena in which microfilaments play a critical role, including chemotaxis, cell locomotion, phagocytosis, cytokinesis, and the formation of cell surface projections. To perform many of these cellular functions, actin filaments must be intimately associated with the plasma membrane. Determining how actin filaments interact with the membrane will, for example, help to elucidate how cancer cells metastasize. The intestinal microvillus shows clearly visible links which connect a core bundle of actin filaments to the membrane, the identify of this tip component is completely unknown. Helically arranged lateral links extending to the membrane are visible down the length of the microvillar core; they are comprised of a 110 kD polypeptide complexed with calmodulin. Study of the 110K-calmodulin complex can provide needed insight into how actin filaments are anchored. Binding of 110K-calmodulin to F-actin is ATP, calcium, and protein concentration dependent and exhibits cooperativity. In addition, the 110K-calmodulin complex resembles myosin in its ability to decorate actin filaments and to hydrolyze ATP raising speculation that it is a motile molecule in non-muscle cells. The goal of this study is to characterize the interaction of the cytoskeleton with the membrane. In particular, we will: (a) investigate whether purified 110K- calmodulin will interact directly with lipid, (b) use cross-linking experiments on intact microvilli to probe for a protein which may serve to link 110K-calmodulin to the membrane, followed by characterization of this linker protein, (c) determine the components which comprise the microvillar tip region, (d) search for the presence of 110K-calmodulin proteins in other tissue types, and (e) continue to map the functional domains of the 100 kD polypeptide including identification of the ATP-binding site, actin- binding site, and calmodulin-binding site within the molecule.