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.     

Yeast actin-binding proteins: evidence for a role in morphogenesis

Three yeast actin-binding proteins were identified using yeast actin filaments as an affinity matrix. One protein appears to be a yeast myosin heavy chain; it is dissociated from actin filaments by ATP, it is similar in size (200 kD) to other myosins, and antibodies directed against Dictyostelium myosin heavy chain bind to it. Immunofluorescence experiments show that a second actin-binding protein (67 kD) colocalizes in vivo with both cytoplasmic actin cables and cortical actin patches, the only identifiable actin structures in yeast. The cortical actin patches are concentrated at growing surfaces of the yeast cell where they might play a role in membrane and cell wall insertion, and the third actin-binding protein (85 kD) is only detected in association with these structures. This 85-kD protein is therefore a candidate for a determinant of growth sites. The in vivo role of this protein was tested by overproduction; this overproduction causes a reorganization of the actin cytoskeleton which in turn dramatically affects the budding pattern and spatial growth organization of the yeast cell.     

SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.     

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.     

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.     

Colocalization of F-actin and 34-kilodalton actin bundling protein in Dictyostelium amoebae and cultured fibroblasts

The Ca2+-sensitive actin-binding protein isolated from Dictyostelium discoideum, 30,000-D protein (Fechheimer and Taylor: J. Biol. Chem. 259:4514-4520, 1984;) has recently been localized in filipodia of substrate-adhered amoebae (Fechheimer: J. Cell Biol. 104:1539-1551, 1987). We have determined that this protein has a Mr of 34,000 daltons and is strictly colocalized with actin filaments in both substrate-attached Dictyostelium amoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34-kilodalton (kD) protein that cross-reacts specifically with antibody to the Dictyostelium bundling protein. Mammalian 34-kD protein is colocalized with F-actin in stress fibers and the cortical cytoskeleton in substrate-adhered fibroblasts. In substrate-adhered vegetative Dictyostelium, F-actin and 34-kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34-kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetative Dictyostelium, F-actin and 34-kD protein were not colocalized in cells that had progressed through the developmental cycle. In fruiting bodies, 34-kD protein was detected by immunofluorescence microscopy only in prespore cells, while F-actin appeared in stalk cells and spores.     

Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH- terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.     

Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625- 14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.     

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.     

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.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).     

Nuclear actin and myosin as control elements in nucleocytoplasmic transport

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.     

Plasma membrane association of Acanthamoeba myosin I

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F- actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI- extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP- sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin- binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.     

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiêm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.     

Secretory cell actin-binding proteins: identification of a gelsolin- like protein in chromaffin cells

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.     

Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.     

Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets

The Mr approximately 540,000 dimeric actin gelation protein, actin-binding protein (ABP), has previously been shown in human platelets to link actin to membrane glycoprotein Ib (GPIb) (Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977; Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321). We have examined further the interaction between ABP and GPIb. Platelet extracts were depleted of ABP by precipitation with anti-ABP monoclonal antibodies (mAbs); in resulting precipitates, ABP monomer is complexed with GPIb in a 5:1 molar ratio. The ABP.GPIb complex is resistant to chaotropic solvents but dissociated by the ionic detergent, sodium dodecyl sulfate. Treatment of intact platelets with the ionophore A23187 activates a Ca2+-dependent protease which cleaves the Mr approximately 270,000 ABP subunit into three fragments of Mr 190,000, 100,000, and 90,000; the latter fragment is derived from the Mr 100,000 fragment. Anti-ABP mAbs coprecipitated GPIb with the Mr 100,000 and 90,000 fragments, but not with the Mr 190,000 fragment which contains the ABP self-association site. In the reciprocal experiment, anti-GPIb antibodies co-precipitated only the Mr 100,000 and 90,000 ABP fragments. Actin also co-precipitated with the Mr 100,000 and 90,000, but not with the Mr 190,000 ABP fragment. The anti-ABP mAb that precipitated the Mr 100,000-90,000 GPIb-binding ABP fragment recognizes a trypsin cleavage fragment of ABP that binds actin filaments in vitro. These findings establish that both the GPIb-binding site and actin-binding sites are in the same region of the ABP monomer. Because of the extended bipolar conformation of the ABP molecule, the data suggest that the GPIb.actin-binding region is located remote from the self-association, or dimerization, site of the ABP subunit.     

Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis

Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.     

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.