Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

Proteolytic enhancement of gelation of ascites tumor cell extracts. Relationship to actin binding protein

Proteolysis of cytoplasmic extracts of sarcoma 180 and MAT-C1 adenocarcinoma ascites cells enhances the rate of gelation. Only high molecular weight polypeptides, including actin binding protein and myosin, are cleaved during the process; actin is not cleaved. In MAT-B1 adenocarcinoma extracts the gelation rate was not enhanced by proteolysis and actin binding protein was not readily cleaved. Electrophoretic comparisons of trypsin-treated and untreated extracts of MAT-B1 and MAT-C1 cells show that actin binding protein is the only readily discernible polypeptide which is cleaved in the C1 cells but not in the B1 cells. These results suggest that actin binding protein may act as an inhibitor of gelation.     

Interactions of cytoskeletal elements with the plasma membrane of Sarcoma 180 ascites tumor cells

The association of cytoskeletal proteins with cell surface envelopes from Sarcoma 180 ascites cells has been studied by several techniques previously used successfully in studying the interaction of spectrin with erythrocyte membranes. By electron microscopy the envelopes exhibit irregular exterior surfaces and the presence of substantial amounts of “fuzz” at the interior surface. Extraction of the envelopes at low ionic strength and alkaline pH fragments the membranes and depletes them of the “fuzz” with concomitant elution of four major polypeptides of mol. wt >300 000 (Band E), 250 000, 100 000 and 43 000 D. The last three of these have been tentatively identified as actin-binding protein (ABP), α-actinin and actin. Membrane-associated myosin is not eluted under these conditions. Neither actin nor myosin is eluted under conditions commonly used to depolymerize them. However, myosin can be eluted at high salt concentrations if the envelopes have been previously extracted and fragmented with alkaline buffer as above. Extraction of the envelopes with Triton X-100 removes 60% of the membrane lipid and 70–80% of lactoperoxidase-iodinated cell surface proteins without removal of significant amounts of the cytoskeletal proteins. The Triton residues maintain the shape of the original envelopes but have lost the trilaminar membrane structure. Proteolysis of intact envelopes with trypsin or papain cleaves the high molecular weight polypeptides in the order E > ABP > myosin. Fragmentation occurs with cleavage of E or ABP, but does not appear to require cleavage of myosin. Actin and α-actinin are not appreciably cleaved when associated with the membrane. The results, combined with previous observations, suggest an extensive complex of cytoskeletal proteins attached to the membrane interior surface.     

Calcium sensitivity of hybrid complexes of muscle myosin and Physarum proteins.

1. A myosin-actin hybrid complex was used to study actin-associated calcium sensitivity of a "cytoplasmic" actomyosin. The approach should be generally applicable. 2. Low salt extracts of Physarum polycephalum contain actin which remains in solution after centrifugation at 46 000 times g or at 100 000 times g for 1 h. The actin was precipitated by the addition of muscle myosin to the supernatants and detected in the hybrid complex by electron microscopy, sodium dodecyl sulfate gel analysis, super-precipitation and activation of the myosin ATPase activity. Actin was also precipitable from high speed supernatants of brain tissue or platelets. 3. The hybrid complexes from Physarum possessed 1.5-5-fold calcium dependency which could be removed by washing. Reincubation of the washed complex with concentrated wash solution resulted in high calcium sensitivity. On sodium dodecyl sulfate gels, unwashed complexes from Physarum contained high molecular weight material in addition to bands of molecular weights less than actin. The bands in the size range of 39 000 to 18 000 were primarily lost from the Physarum complex concomitantly with loss of calcium dependence. 4. When the Physarum supernatants were made 40 mM in MgCl2, precipitates were formed containing actin which possessed calcium sensitivity which was also lost on washing with low ionic strength solutions. This calcium dependency was partially reversed by the addition of desensitized rabbit actin to the precipitate before assay. 5. Conclusion: calcium regulation of actomyosin in Physarum is mediated primarily by factors that are bound to the actin component. The regulatory factors are soluble in low salt buffers. The molecular weights of the polypeptide chains of several of these factors are similar to those of the troponin polypeptides of striated muscle. In Physarum but not in platelet or brain a prominent polypeptide chain of approx. 55 000 molecular weight also occurs which coprecipitates with the hybrid complex and which is not easily removed.     

Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod.

Gelation factor (ABP120) is one of the principal actin-cross-linking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue repeats that have an Ig fold. The ability to dimerize is crucial to the actin cross-linking function of gelation factor and is mediated by the rod in which the two chains are arranged in an antiparallel fashion. We report the 2.2 A resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, and domains 1-5 do not pair with domains from the other chain. This information allows construction of a model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact.     

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.     

Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors

We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.     

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.     

Electron microscopic visualization of the filament binding mode of actin-binding proteins

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.     

Isolation of a new actin-gelling protein from liver

An F-actin crosslinking protein, tentatively named gelactin, has been isolated from rabbit liver. This protein is a dimer of 200 000 molecular weight which promotes the lateral association of actin filaments. The effect is maximal at a molar ratio of gelactin:actin of about 1:1000. The dependency of the gelation process on various experimental factors was studied by a centrifugation assay. The results demonstrate that gelation is greatly influenced by pH, ionic strength and temperature, but is insensitive to the presence of calcium ions. We show that, in our conditions, the association of actin with gelactin does not interfere with actin-myosin interactions and does not influence the kinetics of actin polymerization. The amino acid composition of gelactin is also given. From its overall properties gelactin is shown to be different from all other actin-gelling proteins.     

Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the cAMP-dependent protein kinase in bovine platelets.

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.     

Probe diffusion in cross-linked actin gels

The diffusion of monodisperse polystyrene latex spheres (PLS) in column-purified 0.65 mg/mL actin solutions, polymerized with 100 mM KCl in the absence and presence of a cross-linker, actin binding protein (ABP), has been studied using dynamic light scattering. Measurements over a wide range of scattering angles from 90 degrees to 8 degrees, corresponding to inverse scattering vector probing distances of about 40-400 nm, respectively, give a measure of both the fraction of PLS mobile over the probing distance (from the normalized time autocorrelation function amplitude) and the average diffusion coefficient of the mobile PLS. Both 100- and 500-nm diameter PLS are fully mobile in polymerized actin solutions over distances of less than 100 nm, as reported previously (Newman, J., Schick, K. S. & Gukelberger, G. Biophys. J. 53, 573a, and Newman, J., Mroczka, N. & Schick, K. L. Biopolymers, 28, 655-666). At increasing probing distances, or when ABP is added at molar ratios of 1:750 or 1:150, greater fractions of the PLS are immobilized, up to almost 99% at the conditions of a 400-nm probing distance with 500-nm probes and at a ratio of 1:150 added ABP to actin. The degree of immobilization correlated well with the amount of added ABP, the size of the PLS, and the probing distance. At increasing probe distances, as the degree of immobilization increases, the remaining mobile fraction of PLS has an increasing average diffusion coefficient. These results suggest a range of pore sizes in the actin gels with a mean size of a few hundred nanometers.     

Evidence that a 27-residue sequence is the actin-binding site of ABP-120

Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.     

Isolation of Sugar Compounds of Asparagine,Phenylalanine, Tyrosine and Valine from Cured Tobacco Leaves

Scanning electron microscopy and rigidity characterization were used in this study of myosin gelation in the presence of actin. The heat-induced gel formation of myosin was aided by the addition of actin to myosin at a molar ratio of 1: 2.7. However, this actin effect was not observed when the actin-binding site(s) of myosin was blocked by p-chloromercuri-benzoate treatment or denaturation, or when the myosin-binding site(s) of actin was blocked by trinitrophenylation.

The effect also disappeared with an aging myosin-actin mixture, with ATP or pyrophosphate as dissociating agent prior to thermal treatment of the system.

The present findings indicate the indispensability of myosin binding to actin for enhancing the thermal gelation of myosin. The gelation process appears to involve a large electrostatic contribution, as well as such chemical reactions as oxidation of SH groups.     

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.     

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X- 100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.     

Viscoelasticity of F-actin measured with magnetic microparticles

Dispersed submicroscopic magnetic particles were used to probe viscoelasticity for cytoplasm and purified components of cytoplasm. An externally applied magnetic field exerted force on particles in cells, in filamentous actin (F-actin) solutions, or in F-actin gels formed by the addition of the actin gelation factor, actin-binding protein (ABP). The particle response to magnetic torque can be related to the viscoelastic properties of the fluids. We compared data obtained on F-actin by the magnetic particle method with data obtained on F-actin by means of a sliding plane viscoelastometer. F-actin solutions had a significant elasticity, which increased by 20-fold when gels were formed by ABP addition. Both methods gave consistent results, but the dispersed magnetic particles indicated quantitatively greater rigidity than the viscoelastometer (two and six times greater for F-actin solutions and for F-actin plus ABP gels, respectively). These differences may be due to the fact that, compared with traditional microrheometers, dispersed particle measurements are less affected by long-range heterogeneity or domain-like structure. The magnetometric method was used to examine the mechanical properties of cytoplasm within intact macrophages; the application of the same magnetometric technique to both cells and well-defined, purified protein systems is a first step toward interpreting the results obtained for living cells in molecular terms. The magnetic particle probe system is an effective nonoptical technique for determining the motile and mechanical properties of cells in vitro and in vivo.     

Interaction of filamentous actin with isolated liver plasma membranes

Actin-membrane interactions have been studied using purified liver plasma membranes and muscular filamentous actin. Despite the large quantity of endogenous actin present in membranes, exogenous muscular filamentous actin cosediments with membranes after a 30 min centrifugation at 30 000 g. The cosedimentation process is time-dependent and exhibits a complex relationship with actin concentration. The cosedimentation of actin with membranes can be partly explained by gelation as shown by low-shear viscosity and electron microscopy. The characterization of the gelation phenomenon as a function of time, actin and membrane concentrations, ionic strength, temperature and Ca2+ concentration is also presented. Gelation alone cannot however account for the overall cosedimentation data, and a more direct mode of association between actin and the membrane must be envisaged. The analogy that exists between the results obtained with liver plasma membranes and those obtained with other membrane systems suggests that a general mechanism may be involved in the interaction of actin with plasma membranes.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.