Mapping of the microvillar 110K-calmodulin complex: calmodulin- associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity

The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the 110K-calmodulin complex with alpha-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actin-binding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrow-head appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium- or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at approximately 40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with alpha-chymotrypsin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATP. Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the 110-kD polypeptide. The 78 kD, two of the three fragments at approximately 40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the 110K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.     

Reassociation of microvillar core proteins: making a microvillar core in vitro

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross-bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K-calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K-calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K-calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.     

Isolation and partial characterization of a 110-kD dimer actin-binding protein

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.     

Characterization of intestinal microvillar membrane disks: detergent- resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin)

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.     

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

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding 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.     

Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion dependent conformational change

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unknown function [Coluccio, L. M., & Bretscher, A. (1988) J. Cell Biol. 106, 367-374]. By analysis of limited alpha-chymotryptic cleavage products, we now show that the molecular organization is very similar to that described for the S1 fragment of myosin. The catalytic site was identified by photoaffinity labeling with [5,6-3H]UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by [5,6-3H]UTP. Physical characterization revealed that the 45- and 32-kDa fragments exist as a complex that can bind F-actin, whereas the 40-kDa/32-kDa complex cannot bind actin. We conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the microvillar 110-kDa calmodulin complex (myosin-1) in kidney.

The epithelial layer lining the proximal convoluted tubule of mammalian kidney contains a brush border of numerous microvilli. These microvilli appear in structure to be very similar to the microvilli on epithelial cells of the small intestine. Microvilli found in both the small intestine and the proximal convoluted tubules in kidney have a core bundle of actin filaments bundled by the accessory proteins villin and fimbrin. Along the length of intestinal microvilli, lateral links can be observed to connect the core bundle of actin filaments to the membrane. These cross-bridges are comprised of a 110-kDa calmodulin complex which belongs to a class of single-headed myosin molecules, collectively referred to as myosin-1. We now report that an analogous calmodulin-binding polypeptide of 105 kDa has been identified in rat kidney cortex. The 105-kDa polypeptide is preferentially found in purified kidney brush borders, can be extracted with ATP, and co-elutes with calmodulin on gel filtration and anion exchange chromatography. Fractions containing the 105-kDa polypeptide exhibit a modest ATPase activity in buffer containing CaCl2. The partially purified 105-kDa polypeptide will bind iodinated calmodulin and will sediment with F-actin in buffer containing ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+. The addition of ATP partially reverses this association with F-actin. These results indicate that myosin-1, in addition to its presence in intestinal brush borders, is present in the brush border of kidney. We also provide preliminary evidence to indicate that the 105-kDa polypeptide is not restricted to tissues possessing a brush border.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme

The 110-kD protein-calmodulin complex (110K-CM) of the intestinal brush border serves to laterally tether microvillar actin filaments to the plasma membrane. Results from several laboratories have demonstrated that this complex shares many enzymatic and structural properties with myosin. The mechanochemical potential of purified avian 110K-CM was assessed using the Nitella bead motility assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature (Lond.). 303:31-35). Under low Ca2+ conditions, 110K-CM-coated beads bound to actin cables, but no movement was observed. Using EGTA/calcium buffers (approximately 5-10 microM free Ca2+) movement of 110K-CM-coated beads along actin cables (average rate of approximately 8 nm/s) was observed. The movement was in the same direction as that for beads coated with skeletal muscle myosin. The motile preparations of 110K-CM were shown to be free of detectable contamination by conventional brush border myosin. Based on these and other observations demonstrating the myosin-like properties of 110K-CM, we propose that this complex be named "brush border myosin I."     

Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.     

IQGAP1, a Rac- and Cdc42-binding Protein, Directly Binds and Cross-links Microfilaments

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two ~190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D–sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.     

The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase

The microvillus 110-kD protein-calmodulin complex (designated 110K-CM) shares several properties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, 110K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K+ and EDTA (0.24 mumol P1/mg per min) or in the presence of Ca++ (0.40 mumol P1/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 mumol P1/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of 110K-CM is also enhanced by the addition of 5-10 microM Ca++, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca++. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of 110K-CM's enzymatic activity in the presence of 5-10 microM Ca++ and actin. 110K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified 110K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the 110K-CM complex is functionally analogous to the mechanoenzyme myosin.     

Ca2+-calmodulin-dependent regulation of F-actin-myelin basic protein interaction

Myelin basic proteins (MBP) interacts with F-actin resulting in the precipitation of a complex of both proteins. Electron microscope observations of this complex reveal the presence of ordered bundles of F-actin filaments similar to those obtained from F-actin and troponin I. In addition to the bundles, there also appear short fragments of F-actin filaments. In the presence of Ca2+ calmodulin causes a release of MBP from its complex with F-actin, accompanied by dissociation of F-actin bundles into separate filaments. Parallel to the binding of MBP to F-actin the ATPase activity of actomyosin is progressively reduced. This inhibition is reversed by calmodulin but only in the presence of Ca2+. Studies of the binding of S-1 to F-actin and to the F-actin-MBP complex indicate that the interaction sites for MBP and S-1 on the actin molecule are different.     

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells

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 Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) 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.     

Assembly of the brush border cytoskeleton: changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine

The assembly of the intestinal microvillus cytoskeleton was examined during the differentiation of enterocytes along the crypt-villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (hc) of brush border (BB) myosin I (110K-calmodulin complex) and rhodamine-conjugated phalloidin as a probe for F-actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (hc) did not assume this localization until the crypt-villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt-villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane cytoskeleton as well. The pattern of expression of the microvillar core proteins along the crypt-villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.     

Pig thyroid spectrin. A membrane-associated protein related in structure and function to brain spectrin

Thyroid spectrin has been obtained pure from pig thyroid glands. This protein, composed of two non-identical polypeptide chains of 240 kDa and 235 kDa, appears to possess the same structural and immunological properties as well as the same calmodulin and actin-binding properties as brain spectrin. Through cross-linking of actin filaments it is a potent gelation factor for F-actin solutions. It represents one of the major protein of the cytoskeleton underlying the thyroid plasma membrane together with myosin, alpha-actinin and actin.     

Calmodulin dissociation regulates brush border myosin I (110-kD- calmodulin) mechanochemical activity in vitro

110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.     

Identification by monoclonal antibodies and characterization of human platelet caldesmon

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.     

Reevaluation of the hydrophobic nature of the 110-kD calmodulin-, actin-, and membrane-binding protein of the intestinal microvillus

A complex of calmodulin (CM) and the 110-kD (110K) subunit composes the helical array of cross-bridges linking the microvillus actin filament bundle with the membrane. The hydrophobic properties of the 110K protein, assessed by the detergent phase partitioning assay [Bordier C: J Biol Chem 256:1604, 1981], are highly dependent on the solution conditions used in its isolation. The ATP-dissociable 110K-CM complex [Howe and Mooseker: J Cell Biol 97:974, 1983] exhibits hydrophilic characteristics in this assay. In contrast, the 110K subunit extracted from brush borders by Triton X-100, sodium dodecyl sulfate, and sodium pyrophosphate (detergent-treated 110K) [Glenney JR, Glenney P: Cell 37:743, 1984] behaves as a hydrophobic protein. However, because the soluble hydrophilic 110K-CM can be rendered hydrophobic by treating the complex with the same detergent and salt conditions used in the preparation of detergent-treated 110K, the properties of detergent-treated 110K seem likely to be an effect of the solution conditions on its native conformation, sedimentability, or exposure of binding domains. In addition, the detergent-treated 110K is devoid of calmodulin and no longer exhibits the actin-binding activity characteristic of the ATP-dissociable 110K-CM and of the intact complex in situ. With two partially purified preparations of the 110K subunit exhibiting such dramatically distinct properties, it seems premature to define the nature of the 110K subunit's association with the membrane at this time.     

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.