The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.     

Examination of actin polymerization and viscosity induced by cations and ionic strength when cross-linked by alpha-actinin

Increasing potassium chloride concentration from 0 to 100 mM and magnesium chloride from 0 to 2 mM show a parallel rate increase in polymerizing actin, whereas increasing calcium chloride concentration from 0 to 0.2 mM decreases the rate of polymerizing actin. The presence of alpha-actinin has little influence on the polymerization kinetics of actin under these conditions. Viscometric measurements indicate that the presence of various mono- and divalent cations, ionic strength, and alpha-actinin in combination are responsible for changes in the mechanical properties of solutions containing actin. The actin filament dynamic behavior is drastically reduced under these conditions as confirmed by quasi-elastic light scattering.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex

The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.     

Preparation and polymerization of skeletal muscle ADP-actin

Skeletal muscle ADP-G-actin was prepared from ADP-F-actin, which had been freed of residual ATP by repeated sonication, by depolymerization in 5 mM Tris-HCl, 0.2 mM ADP, 0.2 mM dithiothreitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.01% NaN3, pH 8.0. The ADP had been freed of traces of ATP by DEAE-chromatography, and 5 microM diadenosine pentaphosphate was added to inhibit myokinase activity. The kinetics of the spontaneous polymerization of ADP-actin in 1 mM MgCl2 + 0.1 M KCl were compatible with the simple nucleation-elongation model previously used to explain the polymerization of ATP-actin. The critical concentrations of ADP-actin were 8.0 and 2.0 microM in 1 mM MgCl2 and 1 mM MgCl2 + 0.1 M KCl, respectively. These values are 20-30-fold higher than the corresponding values in ATP. Using cross-linked actin trimers to nucleate polymerization, the association rate constants were found to be 0.8 and 0.9 microM-1 S-1 in MgCl2 and MgCl2 + KCl, respectively, which are 0.4 and 0.2 times the values for ATP-actin. The dissociation rate constants, calculated from the critical concentrations and the association rate constants, were 6.4 and 1.8 S-1, respectively, which are 10 and 5 times the corresponding values for ATP-actin.     

Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'- phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.     

Reinvestigation of the inhibition of actin polymerization by profilin

In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).     

Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2

Acanthamoeba profilin strongly inhibits in a concentration-dependent fashion the rate and extent of Acanthamoeba actin polymerization in 50 mM KCl. The lag phase is prolonged indicating reduction in the rate of nucleus formation. The elongation rates at both the barbed and pointed ends of growing filaments are inhibited. At steady state, profilin increases the critical concentration for polymerization but has no effect on the reduced viscosity above the critical concentration. Addition of profilin to polymerized actin causes it to depolymerize until a new steady-state, dependent on profilin concentration, is achieved. These effects of profilin can be explained by the formation of a 1:1 complex with actin with a dissociation constant of 1 to 4 microM. MgCl2 strongly inhibits these effects of profilin, most likely by binding to the high-affinity divalent cation site on the actin. Acanthamoeba profilin has similar but weaker effects on muscle actin, requiring 5 to 10 times more profilin than with amoeba actin.     

Snake venom cardiotoxin induces G-actin polymerization

Snake venom cardiotoxin showed the ability to induce polymerization of G-actin from rabbit skeletal muscle in a low ionic strength buffer composed of 0.2 mM CaCl2/0.2 mM ATP/0.5 mM mercaptoethanol/2.0 mM Tris-HCl, pH 8.0. The activity was enhanced greatly when 0.4 mM MgCl2 was present in the buffer and could be inhibited if G-actin was preincubated with deoxyribonuclease I. Furthermore, the DNAase could also partially depolymerize actin polymer previously formed by the interaction of G-actin with the toxin.     

Interaction of annexin VI with actin

Actin polymerization was investigated using fluorescence probe N-(1-pyrenyl)iodoacetamide, which was bound covalently to reactive sulfhydryl group, Cys-373. Labeled actin in the bulk was 0.5 to 1% of total actin concentration. Actin polymerization at concentration 12 mM was started by addition of 20 mM KCl and 2 mM MgCl2. The label fluorescence was excited at 365 nm and registered at 386 nm. Under actin polymerization the label fluorescence increased almost 10 times. Two main phases may be distinguished in the process of actin polymerization: 1) monomer activation and nucleus (trimer) formation, 2) growth of actin filaments on the nuclei. In our experimental conditions, both for pure actin and for that with added annexin VI, the 1st phase continued for about 3 min and after that the 2nd phase was perfectly approximated by exponential dependence. An analysis of the exponential curves showed that actin monomer lifetime increased from 327 s, at annexin absence, to about 373 s at 0.7 microM annexin and more. Calculation of rate constants at two ends of growing actin filament suggests that annexin VI binds with pointed ("slow") end so that at sufficient annexin concentration the filament grows only on barbed ("fast") end. Our results, together with data of other researchers showing that annexin VI binds with the inner membrane surface of smooth muscle cell through Ca2+, may indicate that, at Ca2+ entering the cell, this annexin binds actin filament pointed ends to cell surface making it ready for the act of contraction.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.     

Effect of erythrocyte spectrin on actin self-association

The polymerization of pyrene-labelled skeletal muscle actin has been monitored in the presence of chromatographically purified spectrin dimers and tetramers. A small but consistent effect of spectrin binding on the critical concentration was observed for actin polymerized in the presence of 1 mM MgCl2. These data were analysed using the principle of linked functions. Spectrin binds exclusively to the filamentous form of actin, and thereby stabilizes F-actin with respect to the G-form. The decrease in the critical concentration for actin polymerization, in the presence of spectrin, has been shown to be consistent with an equilibrium constant for the binding of spectrin to individual promoters within F-actin of approximately 8 X 10(5) M-1 at 23 degrees C, and an ionic strength of 7 mM.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

The formation of actin oligomers studied by analytical ultracentrifugation

The small oligomers formed from Mg-G-actin under favorable conditions were studied by sedimentation velocity ultracentrifugation. The critical concentration of actin at pH 7.8 in the presence of 100 microM MgCl2 and 200 microM ATP was 12.5 +/- 2.8 microM. Under these conditions, about 15% of 7.5 microM Mg-actin was converted to oligomers of subunit size four to eight in 5 h at 20 degrees C. In 100 microM MgCl2 and no free ATP, the critical concentration was about 6.5 microM, and about 22% of 7.5 microM Mg-actin was converted to dimers in 80 min. There were no detectable higher oligomers or F-actin present in either case. As determined by the analysis of ATP hydrolysis, most, if not all, of the oligomer subunits contained ATP. When 28.5 microM actin was polymerized to steady state in 100 microM MgCl2 and 200 microM ATP, about 50% of the actin was present as F-actin, consistent with the critical concentration (approximately 12.5 microM), about 50% as oligomers as large as seven subunits, and only about 5% as monomers. When solutions containing oligomers were diluted the oligomers dissociated. Alternatively, when the MgCl2 concentration was raised to 1 mM, the solutions containing oligomers polymerized more rapidly than monomeric Mg-G-actin and to the same final steady state. These data are entirely consistent with the condensation-elongation model for helical polymerization proposed by Oosawa and Kasai (Oosawa, F., and Kasai, M. (1962) J. Mol. Biol. 4, 10-21) according to which, under certain conditions, substantial amounts of short linear and helical oligomers should be formed below the critical concentration and linear oligomers should coexist with monomers and F-actin at steady state.     

Preparation of membrane-bound and of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity.

The effect of spectrin on the polymerization of muscle actin has been investigated by hydrodynamic methods and electron microscopy. Spectrin markedly accelerated polymerization of actin. The effect was more easily observed in lower concentrations of KCl (e.g. 24 mM) where spontaneous polymerization was negligibly small. Similarly large acceleration was observed for polymerization in MgCl2 or CaCl2. The rate of polymerization of actin was proportionally increased with the concentration of spectrin added to a fixed concentration of action. The stationary level of specific viscosity also increased with the spectrin concentration, but at larger concentrations it became smaller. The flow birefringence and electron microscope measurements indicated that actin polymers formed under the influence of spectrin were shorter than those of control F-actin filaments. The structural viscosity and electron microscope observations suggested that the interaction between F-actin fibers was not increased by spectrin. These data strongly suggest a seeding role of spectrin in the polymerization of actin. Spectrin accelerates formation of the nuclei for polymerization. The more the nuclei are formed, the larger the number of the grown polymers are and this leads to rapid formation of shorter polymers since the amount of actin is limited. The acceleration activity was found only in freshly prepared spectrin from fresh ghosts taken from freshly drawn blood.     

Changes in actin lysine reactivities during polymerization detected using a competitive labeling method

We have studied the structure of actin by measuring the relative reactivities of lysines with acetic anhydride using a competitive labeling procedure comparing monomeric globular actin. monomeric actin in the presence of salt, and filamentous actin polymerized in 100 mM NaCl and 100 mM NaCl, 2 mM MgCl2. We have identified 12 of the 19 lysines: 18, 50, 61, 68, 113, 191, 237, 290, 315, 325, 327, and 358. In all conditions, Lys (325, 327) is the most reactive. In globular actin, Lys 18, 191, 290, 314. and 358 are less than 20% as reactive as Lys (325, 327); the remaining have intermediate reactivities. On polymerization in the presence of NaCl and Mg2+, lysines 50, 61, 68, 113, and 290 become less reactive relative to Lys (325, 327). The changes in Lys 50, 61, and 113 are due largely to the polymerization event whereas those in Lys 68 and 290 appear to be an effect of Mg2+. Lys 18, 191, and 358 increase in relative reactivity when cation is added to the monomer and then become less reactive in the polymer, showing no large overall change in reactivity relative to the monomer in the absence of salt. Lysines that are reduced in reactivity upon polymerization indicate possible contact regions between actin monomers in the filament in the NH2-terminal third of the protein.     

Conformational changes in actin resulting from Ca2+/Mg2+ exchange as detected by proton NMR spectroscopy

Skeletal muscle actin can be maintained in a monomeric form in very low ionic strength solutions as well as in high concentrations (0.6 M) of MgCl2 or CaCl2. 400-MHz 1H-NMR spectra revealed characteristic changes which show that the conformation of actin alters by exchanging Ca2+ for Mg2+ in the single high-affinity cation binding site. When all low-affinity cation binding sites are filled (in the presence of high concentrations of Ca2+ or Mg2+), the spectra show that actin conformation differs from that in low-ionic-strength buffer. A comparison of actin in 0.6 M CaCl2 and 0.6 M MgCl2 revealed that the environment of only a small number of protons is affected by the exchange. A new proposal for the essential steps involved in actin polymerization is presented.     

Interaction of maleimidobenzoyl actin with myosin subfragment 1 and tropomyosin-troponin.

A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl (pH 7.6). The resulting polymer (MBS-P-actin) is highly potentiated so that it activates the Mg(2+)-ATPase of S1 more strongly than native F-actin. The affinity of MBS-P-actin for S1 in the presence of ATP (KATPase) is about four times higher than that of native F-actin, although the maximum velocity at infinite actin concentration (Vmax) is almost the same. This high activation is not due to a cross-linking between MBS-P-actin and the S1 heavy chain, since no substantial amount of cross-linking was observed in SDS gel electrophoresis. Direct binding studies and ATPase measurements showed that the modification of actin with MBS impairs the binding of tropomyosin. Tropomyosin binding can be improved considerably by the addition of troponin. However, the regulation mechanism of the acto-S1 ATPase activity by troponin-tropomyosin is damaged. The addition of troponin-tropomyosin reduces the S1 ATPase activation by MBS-P-actin to the same level as that of native F-actin in 30 mM KCl/2.5 mM ATP/2 mM MgCl2, but there is no difference in the ATPase activation in the presence and absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)