Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells.

Abstract

One of the major proteins of the chicken intestinal microvillus is a calmodulinbinding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane-intact brush borders with ATP and obtained solubilization of the 110-kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP-induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein:CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 x 8 nm to 8 x 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium-independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P. T., and D. R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.Interactions between actin and membranes are an almost universal organizational feature of cortical cytoplasm in eucaryotic cells. Such interactions can be involved in establishment or maintenance of cell shape as well as in movements of the cell. Ultrastructural studies have shown that actin is localized near the plasma membrane in many cell types, with apparent attachments between actin filaments and the membrane. (For a review of membrane-cytoskeletal interactions, see references 52.) An excellent model for investigating how actin filaments interact with membranes is the brush border of intestinal epithelial cells. Each microvillus of the brush border contains an actin filament bundle connected both at its tip and along its length to the surrounding membrane. (For a review of brush border structure, see reference 34.) At the tip of th...

Biosynthesis of microvillar proteins

“…Bundling of the 20 or so actin filaments that are found in each intestinal microvillus probably occurs with the aid of villin, an actin-binding protein, and fimbrin (Matsudaira et al, 1983). The folding of the lipid membrane occurs by cross-linking of the actin filaments to a membrane protein(s) via the 110 kDa protein-calmodulin complex (Howe & Mooseker, 1983). A 140kDa protein, recently purified and characterized , is implicated as the membrane protein participating in this folding/microvillar-elongation process.…”

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

“…The ll0-kD protein is a main component of the lateral bridges that connect the core bundle to the surrounding plasma membrane (13,31,37,46,47,69). Two recent studies indicate that the 110-kD protein shares several properties of a myosin-like mechanoenzyme (12,14).…”

“…With respect to the ll0-kD protein, there is increasing evidence that it shares several properties of a myosin-related mechanoprotein (12,14). This has led to the suggestion that the ll0-kD protein may not only link the core bundle to the plasma membrane (13,31,37,46,47) but may also connect vesicles to the rootlet bundles and thus may play a role in transport of vesicles through the terminal web (14).…”

“…Each microvillus is supported by an axial bundle of actin filaments that are in parallel alignment and display identical polarity (51,53,59; for review see references 8,52). The microvillus core bundle is attached to the surrounding membrane by periodically arranged lateral bridges that consist of a complex of a rod-shaped ll0-kD protein and calmodulin (13,31,37,46,47,69). The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2,4,5,6,25,55).…”

Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

“…Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPllb and III exist: one that binds directly to endogenous membrane actin and one that does not.Numerous studies have provided largely indirect evidence for transmembrane interactions among surface proteins and receptors of cells and an internal membrane matrix composed of actin and other cytoskeletal proteins (3,5,7,10,14,16,17,19,23,25,27,31,35,40,45). Indeed, a number of studies have shown that the surface topography and lateral mobility of receptors can be modulated by the underlying cytoskeleton and its associated contractile and motile elements (20,28,30,31,36,37,39).…”