The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L ., M . S. Mooseker, and T. A. Graves . 1980 . Brush-border calmodulin : a major component of the isolated microvillus core . J. Cell ) that the addition of Ca" (>10 -s M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle . High-speed centrifugation of microvillus cores treated with Ca" indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95-and 70-kdalton subunits . Gel filtration of such Ca" extracts in the presence and absence of Ca" indicates that microvillar actin "solated" by Ca" is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca" results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca" extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca" . In the presence, but not absence, of Ca", 95K inhibits actin assembly (50% inhibition at 1 :50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin-95K complex formed in the presence of Ca" is pelletable after long-term centrifugation . In the absence of Ca", 95K cosediments with F-actin . EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca" . Reassembly of filaments occurs once Ca ++ is removed. In the absence of Ca", 95K has no effect on filament structure and, at relatively high ratios (1 :2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.Studies on the structure, chemistry, and contractility of the brush border ofintestinal epithelial cells have been among the most convincing for establishing basic similarities in the organization and function of contractile proteins in muscle and nonmuscle cells (2, 3, 13, 18-20, 22, 23, 26, 27, 30-32) . Nevertheless, recent work on the structure and chemistry of the brush-border microvillus has led us to hold serious doubts regarding a simple, sarcomere model (19,20,26,30) for the functional organization of actin and myosin in the brush border . We have shown that the addition of Ca" to isolated microvillus cores results in a drastic but at least partially

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...

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle . Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure . The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons . The supernate from the brushborder preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons . The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca", and (c) 4-5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca" . With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin . In the presence of Ca" (>5 X 10-' M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable form. The dissociation of calmodulin was reversed by removal of Ca" . If microvillus cores were pretreated with phalloidin, the Ca"-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1 :10 in the demembranated brush border and 1 :2-3 in the microvillus core . No calmodulin was detected in the detergent-solubilized brush-border membrane fraction .Movements of microvilli on the apical, brush-border surface of cells lining the intestine may facilitate the absorptive functions of this epithelium . In vivo observations of brush-border motility have been recorded (32, 38), although adequate documentation of these technically difficult observations is still lacking . Studies on the isolated brush border indicate that this organelle is a highly organized, motile apparatus comprised of actin, myosin, and various associated proteins that are organized in a functional configuration analogous to that determined for actin and myosin in striated muscle (5,(25)(26)(27) . In vitro experiments on contractile models of the brush border have helped to 916 reveal possible molecular mechanisms for microvillar movement (25,30) . The addition of MgATP and micromolar levels of free calcium ions to demembranated brush borders isolated from chicken intestine causes rapid retraction of the microvillar cores into and through the terminal web (25). Presumably, this contractile mechanism coupled to a relaxation mechanism, perhaps involving the extensive linkages of the microvillus cores to the plasma membrane (6,24,25,27,28), is responsible for cyclic movements of microvilli in vivo.We hope to determine the molecular basis for the observed calcium sensitivity of this contrac-J . CELL BIOLOGY

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