Isolation and characterization of actin and actin-binding protein from human platelets

Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.     

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Ca²⁺-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10^?6–10^?7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10^?7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.     

Effect of tropomyosin on the interactions of actin with actin-binding proteins isolated from pig platelets

Several non-muscle tropomyosins have been reported to lack the ability to polymerize in a head-to-tail manner [Dabrowska, R. et al. (1983) J. Muscle Res. Cell Motil. 1, 83-92; C?té, G.P. (1983) Mol. Cell. Biochem. 57, 127-146]. Unlike rabbit skeletal muscle tropomyosin, these proteins could therefore not protect the F-actin microfilaments neither from disassembly or from cross-linking by the other actin-associating factors. However, we have provided evidence that, in vitro, pig platelet tropomyosin, although shorter in molecular length, exhibits the same properties as the muscle protein: it self-associates and forms a 1:6 complex with platelet filamentous actin under physiological conditions [Prulière et al. (1984) J. Muscle Res. Cell Motil. 6, 126]. In this paper, we examine the effects of several other actin-binding proteins on the microfilaments saturated with this non-muscle tropomyosin. Since contractile proteins often vary with the cell type and may require different conditions for their interactions, we have developed a procedure which allows the parallel purification of actin-binding protein (ABP), vinculin, alpha-actinin, gelsolin as well as actin and tropomyosin from the same batch of cells. Thus, using an homogeneous system, we show by viscometry, sedimentation and densitometry, and by electron microscopy, that pig platelet tropomyosin can protect the structure of the microfilaments from the action of the modulating factors to the same extent as rabbit skeletal muscle alpha-tropomyosin. Our data suggest that interaction of ABP, vinculin or alpha-actinin can occur only with the ends of the filaments when F-actin is saturated with tropomyosin, while cross-linking takes place by interactions with sites localized along the entire length of F-actin in the absence of tropomyosin. Moreover, the presence of tropomyosin on F-actin leads to the total inhibition of gelsolin severing activity, although it did not prevent the binding of gelsolin to the F-actin--tropomyosin complex. This suggests that pig platelet as well as skeletal muscle tropomyosins have the ability to increase the strength of the interaction between actin monomers within the filament. This also suggests that the binding sites of gelsolin along the filaments are not localized in the groove of the F-actin helix.     

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.     

Inhibition of plant calcium-dependent protein kinases by basic polypeptides

Wheat embryo Ca2+-dependent protein kinase (CDPK) is inhibited by a variety of polypeptides including actin, gramicidin S, melittin, protamine, various histone preparations, histone H4 and by basic amino-acid homopolymers. Melittin (Ki 9 microM) is a non-competitive inhibitor of wheat germ CDPK and also inhibits wheat leaf CDPK and silver beet leaf CDPKs. Protamine inhibits wheat germ CDPK in an apparently competitive fashion (Ki 0.2 microM) and is also a potent, albeit less effective, inhibitor of the leaf CDPKs. Various basic amino-acid homopolymers are also potent, apparently competitive inhibitors of wheat embryo CDPK, namely poly(L-lysine) (IC50 2 nM), poly(L-ornithine) (IC50 3 nM) and poly(L-arginine) (IC50 17 nM) and also inhibit the leaf CDPKs, albeit at higher concentrations. Histone H4 and various calf thymus histone preparations inhibit wheat embryo CDPK in a fashion that is not competitive and calmodulin can substantially reverse such inhibition.     

Studies on the actin-binding protein HS1 in platelets

Background  

The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/- mice.     

Effect of actin-binding protein on the sedimentation properties of actin

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F- actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Immunoelectron microscopic localization of actin, alpha-actinin, actin-binding protein and myosin in resting and activated human blood platelets

Blood platelets are particularly rich in cytoskeletal proteins and respond to stimulation and activation by changes in shape. We examined the effect of blood platelet activation on the subcellular distribution of the cytoskeletal proteins, actin, myosin, alpha-actinin and actin-binding protein. These studies were performed with immunofluorescent staining on thin cryosections of paraformaldehyde-fixed platelets and by immunogold labeling of ultrathin cryosections of glutaraldehyde-fixed blood platelets. Platelets were studied immediately at blood collection (resting platelets), in platelet-rich plasma and after gel filtration (partially activated platelets), and after gel filtration and thrombin activation (0.5 U/ml, 10 min, 37 degrees C) (activated platelets). Resting platelets were disk-shaped and showed homogeneous distribution of cytoskeletal proteins. Partially activated platelets were more spherical and showed at least one protrusion. Immunofluorescence and immunogold labeling showed a more intense staining of the peripheral 0.2 to 0.3 micron of cytoplasm of these platelets. In the immunofluorescence photographs this resulted in the appearance of small fluorescent rings with staining at the periphery of cross-sectioned cells. Activated platelets showed an irregular outline composed of broad based pseudopods. Cell centers were composed of poorly delineated electron-dense material, interspersed with profiles of surface-connected tubules. The broad based pseudopods stained uniformely for actin, alpha-actinin and actin-binding protein. The cell center stained poorly for these proteins. Myosin staining was found in the peripheral cortex, but also in the cell center. Partially activated platelets that had returned to the disk shape after incubation at 37 degrees C showed increased submembranous concentration of microfilament proteins. These data reveal the profound cytoskeletal rearrangements that already occur upon minimal platelet activation and emphasize that platelets that have returned to the disk shape are not identical to resting platelets.     

Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii

A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.     

Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three- dimensional network resembling the peripheral cytoskeleton of motile cells.     

Low-temperature induction of calcium-dependent protein phosphorylation in blood platelets

Exposure to low temperature causes platelets to change shape in a manner similar to the shape change that precedes secretagogue-induced serotonin release. Previous studies have shown that two proteins, of approximately 20,000 and approximately 40,000 Mr, become phosphorylated before secretion. We have investigated whether low temperature can induce phosphorylation of these proteins and/or serotonin secretion. The data indicate that low-temperature-induced shape change has no requirement for extracellular calcium, whereas phosphorylation of the two proteins and subsequent serotonin release both have strong calcium requirements. Because cold treatment is thought to influence platelet shape through an effect on microtubules, the events in the shape change- release sequence would seem to be ordered as follows: microtubule disassembly leads to shape change leads to protein phosphorylation leads to secretion.     

Modulation of actin polymerization by 47,000-dalton protein of human platelets

We purified 47,000-dalton proteins from both thrombin-stimulated and unstimulated human platelets. The purity of the protein was almost 80% on SDS-polyacrylamide gel electrophoresis. The protein obtained from unstimulated platelets strongly inhibited actin gelation when its molar ratio to actin was 1:200 or higher. The protein obtained from thrombin-stimulated platelets had no inhibitory activity. The results suggest that the 47,000-dalton protein modulates actin polymerization through phosphorylation.     

A 90 000-dalton actin-binding protein from platelets. Comparison with villin and plasma brevin

Affinity chromatography of Ca2+-containing extracts of platelets on DNAase I-Sepharose, using Ca2+-free buffer as eluant, selects a 1:1 complex of a 90 000-dalton protein with actin. The complex shows little interaction with either DNAase or actin unless Ca2+ is present. In the presence of Ca2+, the complex nucleates polymerization of actin, reduces the viscosity attained, and delays filament formation from profilactin with characteristics closely resembling those shown by chicken villin. Proteolysis of the native proteins indicates structural similarity between the platelet protein and villin or villin core; limited proteolytic digestion in the presence of SDS distinguishes the platelet protein from villin but not from the functionally related plasma protein, brevin. The platelet protein is not accessible to enzyme-mediated iodination of surface components on intact cells. The term 'platelet brevin' is proposed for the protein.     

Abundance, relative gelation activity, and distribution of the 95,000- dalton actin-binding protein from Dictyostelium discoideum

We have studied the abundance, relative gelation activity, and distribution of the 95,000-dalton actin-binding protein in Dictyostelium discoideum amoebae. The 95,000-dalton protein was a prominent polypeptide as assessed using quantitative densitometry and radioimmunoassay. We estimated that this protein comprised approximately 1.2% of the protein in a soluble extract of amoebae. The molar ratio of the dimeric 95,000-dalton protein to actin in the soluble extract was 1:30. The apparent viscosities of actin mixtures with either the purified 95,000-dalton protein or the soluble extract were measured by falling ball viscometry in an attempt to assess the contribution of the 95,000-dalton protein to gelation of the soluble extract. The gelation of the soluble extract was significantly less than that expected from the contribution of the 95,000-dalton protein alone. Consequently, we questioned the validity of quantitative analyses of the contributions of specific actin-binding proteins to the gelation of cell extracts. The apparent distribution of the 95,000- dalton protein was observed in chemically fixed and extracted cells by immunofluorescence microscopy and compared with the distribution of cytoplasm and organelles visible using light microscopy. The 95,000- dalton protein was dispersed throughout the cytoplasm of fixed cells, was apparently excluded from prominent organelles, and displayed brightest fluorescence in regions of hyaline cytoplasm. These regions of hyaline cytoplasm that exhibited the brightest fluorescence were observed in the cortical region of rounded cells and in pseudopods of polarized cells. Thus, cell shape and polarity may also have influenced the apparent distribution of the 95,000-dalton protein observed by immunofluorescence microscopy. Study of the distribution of fluorescein- labeled ovalbumin injected into living cells supported the interpretation that the thickness of the cell and the distribution of organelles contributed to the apparent distribution of the 95,000- dalton protein observed in fixed cells using immunofluorescence microscopy. We suggest that the 95,000-dalton protein contributes to modulation of the consistency and contractility of the cytoplasm of D. discoideum amoebae, since it could cross-link actin filaments in vitro in a reversible process that was regulated by changes in the concentration of calcium and of protons, and since it was present in large quantity in the cytoplasm of these cells.     

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.     

On the association of glycoprotein Ib and actin-binding protein in human platelets

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.     

Regulation of water flow by actin-binding protein-induced actin gelatin.

Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation.     

Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

A key event in signal transduction in many eukaryotes is activation of polyphosphoinositide-specific phospholipase C (PIC). This enzyme hydrolyses the plasma membrane-associated lipid, phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P₂) which leads to the production of the two second messenger molecules: inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃) and 1,2-diacylglycerol (DG). In plants, an enzyme which functionally resembles mammalian PIC is known to exist in the plasma membrane, but little is understood about how its activity is regulated. The recent discovery of several plant proteins with 30–40% homology to the mammalian actin- and phosphoinositide-binding protein, profilin, has prompted an investigation as to whether these proteins (plant profilins) are able to interact with polyphosphoinositides and, if so, whether such interactions have physiological relevance for signal transduction via the plant phosphoinositide system.
In this study it is demonstrated that a direct and highly specific interaction does exist between plant profilin and polyphosphoinositides and that these interactions dramatically affect the ability of plant plasma membrane phosphoinositide phospholipase C to utilize phosphoinositides for second messenger production. These data are the first to demonstrate a functional role of plant profilin in controlling polyphosphoinositide turnover and also provide the first evidence for a direct effect of an actin-binding protein on a membrane-associated signalling enzyme. These findings indicate a novel mechanism for control of plant phosphoinositide turnover, and suggest a possible link between plant cell activation, second messenger production and modulation of cytoskeletal dynamics.