High Molecular Weight Actin-binding Protein Research Articles

Actin‐binding protein filamin A is displayed on the surface of human neuroblastoma cells

We previously reported the identification of natural human IgM antibodies, which recognize a M(r) 260 000 surface protein (NB-p260) and induce both complement-mediated cytotoxicity and apoptosis of human neuroblastoma cells. NB-p260 was shown to belong to the family of filamin proteins. Filamin A is a high molecular weight actin-binding protein, previously thought to be only located intracellularly. Here we show that NB cells as well as three NB-unrelated human cell lines express filamin A also on the cell surface. Our findings suggest new biological functions for filamins, including a role as mediators in anti-NB IgM-induced apoptosis, and they add to the growing body of evidence of the interaction of cytoskeletal proteins with the extracellular matrix.

Studies on microplasmodia of Physarum polycephalum: VIII. Visualization of a membrane skeleton and characterization of membrane proteins

Small plasmodial fragments derived from the acellular slime mold Physarum polycephalum were used to demonstrate a functional relationship between ultrastructural changes of the membrane-cytoskeleton complex and rhythmic activities of the motive force generating system. During contraction the microfilament layer detaches from the plasma membrane and moves inwards thus causing syneresis of central granuloplasm from peripheral hyaloplasm. Simultaneously, on the internal face of the plasma membrane a more or less regular two-dimensional network of short filaments and small particles becomes visible resembling the spectrin membrane skeleton of erythrocytes. With incipient relaxation the contracted microfilament system disintegrates and a new layer is formed at the internal cell surface. Biochemical studies on isolated plasma membranes by gel-electrophoresis revealed three distinct bands of 29, 38 and 40 kD as well as some other conspicuous polypeptides such as actin, myosin and, especially, a high molecular weight actin-binding protein (HABP) of 230 kD. Moreover, a 22 kD component was recognized as an integral membrane glycoprotein by Triton X-114 extraction and Con A-peroxidase staining. It is discussed whether the 230 kD and 22 kD proteins are comparable to spectrin and the actin-binding protein ponticulin from Dictyostelium, respectively.

Inositol phospholipid — Induced suppression of F-actin-gelating activity of smooth muscle filamin

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro

The dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freeze-fracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with F-actin in solution) in the circumferential/microfilament bundle.

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18000 M r light chains, while Ca-binding 14000 M r light chains are common to both myosins ( Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022) [15, 16]. We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia ( Anderson et al., Protoplasma 89 (1976) 29 [4]. Antibodies against plasmodial myosin heavy chain (PMHC) and 18000 M r plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells—but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.

Isolation of a 240-kilodalton actin-binding protein from Dictyostelium discoideum.

A high molecular weight actin-binding protein with subunit mass of 240 kilodaltons has been purified from vegetative amoebae of Dictyostelium discoideum. Briefly, a cell extract was prepared by homogenizing vegetative amoebae in 5 mM EGTA, 5 mM 1,4-piperazineethanesulfonic acid, 1 mM dithiothreitol, 0.02% NaN3, pH 7.0, followed by ultracentrifugation at 114,000 X g for 1 h. The 240-kDa protein in this extract was separated from actin by chromatography on ATP-saturated DEAE-cellulose and further purified by chromatography on hydroxylapatite and Sephacryl S-300. The 240-kDa protein increases the low shear viscosity of F-actin. Covalent cross-linking with dimethyl suberimidate demonstrates that the 240-kDa protein can form dimers in high salt (500 mM NaCl). Hydrodynamic studies in high salt demonstrate the presence of an asymmetric dimer (Stokes' radius = 8.6 nm, sedimentation coefficient = 12 S, native molecular weight = 434,000, and frictional ratio = 1.7). Rotary shadowing demonstrates that the monomer is a flexible rod of approximately 70 nm in length that can associate end to end to form a dimer of approximately 140 nm in length. The 240-kDa protein cross-reacts with antibodies to chicken gizzard filamin. The properties of the 240-kDa protein suggest that it is a member of the filamin class of actin-associated proteins.

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins.

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia.

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers.

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.

Further characterization of a brain high molecular weight actin-binding protein (BABP): interaction with brain actin and ultrastructural studies.

Crude actomyosin fraction from porcine brain contained a large amount of high molecular weight actin-binding protein (BABP). The molar ratio of BABP to actin (BABP/actin) in the fraction was estimated to be 0.22. From this fraction, BABP and actin were solubilized with a molar ratio of 0.25, suggesting the existence of an interaction between BABP and brain actin. BABP was finally purified to 90% purity. The purified BABP was negatively stained and observed by electron microscopy; it appeared to be a slender, flexible, two-stranded molecule whose contour length was about 200 nm. The structure was very similar to those of fodrin and other high molecular weight actin-binding proteins such as filamin, spectrin, and ABP. Lattice cage-like structures composed of BABP molecules were occasionally observed at high BABP concentrations. The addition of BABP to actin filaments resulted in the appearance of many branching, filamentous bundles. The electron microscopic observations suggested that a single BABP molecule could crosslink actin filaments, that is, one BABP molecule has two actin binding sites.

A filamin-like protein—a new component of heart myofibrils

A filamin-like protein was identified in cardiac muscle of chick and rat. It was shown that in an extract obtained from chicken cardiac muscle, 1% of the protein had the same M r (250 000–270 000) as the monomer of filamin, a high-molecular weight actin-binding protein from smooth muscle. Immunofluorescent and immunocytochemical analyses of preparations of heart myofibrils and tissue sections revealed that (a) purified antibodies against filamin specifically interacted with heart myofibrils, and (b) the filamin-like protein was localized in the Z line region of the sarcomere.

Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit.

Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.

An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW 260 240 ), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW 260 240 is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW 260 240 is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW 260 240 indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW 260 240 with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW 260 240 and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW 260 240 (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.

Isolation and characterization of a calcium-sensitive alpha-actinin-like protein from human platelet cytoskeletons.

Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization.

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca 2+-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca 2+-activated protease similar to myofibrillar Ca 2+-activated protease has been described in platelets. To explore the role of Ca 2+-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca 2+-activated protease-like activity. The hydrolysis of actin-binding protein by Ca 2+-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca 2+-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca 2+-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca 2+-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E 1, prostaglandin I 2, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca 2+-activated protease in platelets and indicate that the source of calcium important in Ca 2+-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca 2+-activated protease-like activity as measured by the present analytical technique.

Self-association of chicken gizzard filamin and heavy merofilamin

Filamin is a high molecular weight (subunit Mr 250 000) actin-binding protein isolated from smooth muscle. The protein forms a gel when mixed with solutions of F-actin. A proteolytic fragment of filamin, heavy merofilamin (subunit Mr 240 000), generated by the action of Ca2+-activated protease binds to actin but does not produce gelation. We have studied the self-association properties of filamin and heavy merofilamin by direct examination in the electron microscope and by equilibrium sedimentation distribution studies in the ultracentrifuge. Filamin self-associates reversibly to form dimers; the free energy of dimerization is approximately 7 kcal/mol. Further association to form tetramer and multimer appears to be irreversible. Warming of filamin solutions accelerates aggregation. Heavy merofilamin does not appear to self-associate but is entirely monomeric. These studies suggest that filamin produces gelation of F-actin by binding to actin and then self-associating to cross-link actin filaments into a gel.

Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein.

The peripheral cytoplasm of macrophages is involved in the control of locomotion, secretion and endocytosis, events common to many eukaryotic cells. During these activities, the cortical cytoplasm, which contains numerous actin filaments1,2, appears to undergo reversible gel–sol transformations3: cycles of gelation and solation are demonstrable in suitably prepared macrophage extracts, and the gels contain tangled actin filaments4. These changes in consistency of cytoplasmic actin may regulate motile events in the macrophage periphery. Calcium in micromolar concentrations prevents gelation of crude macrophage cytoplasmic extracts4, providing a possible link to abundant indirect evidence implicating calcium in the regulation of locomotion, secretion and endocytosis5. Similar calcium-sensitive gelation phenomena occur in crude cell extracts from diverse cell types and may have a relevance for control of cell movements in general6–11. Actin gelation results from the cross-linking of actin filaments (F-actin) by other proteins. In macrophages, a high molecular weight actin-binding protein (ABP) is the principal actin cross-linking protein12. Cross-linking of actin by these purified actin-binding proteins, however, is insensitive to changes in the calcium concentration4,12, so that another factor must mediate the expression of a calcium effect. We have now isolated such a calcium-dependent regulatory protein from macrophages and call it gelsolin.

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

A high molecular weight protein (HMWP) with properties similar to those of both actin-binding protein (ABP) and filamin has been isolated from cultured baby hamster kidney (BHK-21) cells. The protein was present in an actomyosin-depleted sucrose extract of the cells and was eluted, upon gel chromatography on Sepharose 4B, near the void volume. The subunit migration on sodium dodecyl sulfate/polyacrylamide gels and the amino acid composition of HMWP were similar to those of ABP and filamin. HMWP bound to and crosslinked F-actin from rabbit muscle, as shown by the formation of a gel that was sedimented with low-speed centrifugation. This interaction was insensitive to temperature and low concentrations of calcium ions, although it may depend on the presence of myosin. Observation of thin sections of the actin-HMWP gel revealed crosslinked complexes of laterally aggregated actin filaments. The axial period of the dense crosslinks was 34 nm. The HMWP may be involved in regulation of microfilament organization.

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.