The myosin head can bind two actin monomers

Force impulse is thought to be generated in muscle when myosin head (S-1), while weakly bound to actin filament, undergoes orientational change to form a strong (rigor) bond with actin. There is ample evidence that this bond involves interaction of 1 myosin head with 1 actin monomer. However, X-ray diffraction data of muscle decorated with S-1, as well as recently proposed model of the thin filaments, suggested that each S-1 molecule interacted with two actin monomers. We reinvestigated this controversy and found that the stoichiometry of acto-S-1 bond depended on the relative amounts of actin and myosin present during titrations: when increasing amounts of actin were added to a fixed amount of S-1 (i.e. when myosin heads were initially in excess over actin), the saturating stoichiometry was 1 mol of S-1 per 1 mol of actin. However, when increasing amounts of S-1 were added slowly to a fixed amount of F-actin (i.e. when actin was initially in excess over S-1), the stoichiometry at saturation was 1 mol of S-1 per 2 mols of actin. The ability of S-1 to bind either one or two actin monomers suggests a way that force could be generated during muscle contraction.     

Binding of myosin subfragment-1 to F-actin.

During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.     

Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1

Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.     

Talin binds to actin and promotes filament nucleation

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.     

Cryo-electron microscopy of S1-decorated actin filaments

We have applied techniques for cryo-electron microscopy, combined with image processing, to both S1-decorated native thin filaments and S1-decorated actin filaments. In our reconstruction the actin subunit has a prolate ellipsoid shape and is composed of two domains. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially, the major interaction being with the outermost domain of actin. To distinguish the position of tropomyosin unambiguously in our map, we compared the maps from decorated thin filaments with those from decorated actin filaments. Our difference map clearly shows a peak corresponding to the position of tropomyosin; tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of about 40 Å.As a first step toward looking at the actomyosin structure in a state other than rigor, we examined S1 crosslinked to actin filaments by the zero-length crosslinker EDC in the presence of ATP and after pPDM bridging of the reactive thiols of S1. S1 molecules of the crosslinked complexes in the presence of ATP and after pPDM treatment appear dramatically different from those in rigor. The S1s appear more disordered and no longer assume the characteristic rigor 45° angle with the actin filaments.     

Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy

We have calculated three-dimensional maps from images of myosin subfragment-1 (S1)-decorated thin filaments and S1-decorated actin filaments preserved in frozen solution. By averaging many data sets we obtained highly reproducible maps that can be interpreted simply to provide a model for the native structure of decorated filaments. From our results we have made the following conclusions. The bulk of the actin monomer is approximately 65 X 40 X 40 A and is composed of two domains. In the filaments the monomers are strongly connected along the genetic helix with weaker connections following the long pitch helix. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially and binds to a single actin, the major interaction being with the outermost domain of actin. In the map the longest chord of S1 is approximately 130 A. The region of S1 closest to actin is of high density, whereas the part furthest away is poorly defined and may be disordered. By comparing maps from decorated thin filaments with those from decorated actin, we demonstrate that tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of approximately 40 A. A small change in the azimuthal position of tropomyosin, as has been suggested by others to occur during Ca2+-mediated regulation in vertebrate striated muscle, appears to be insufficient to eclipse totally the major site of interaction between actin and myosin.     

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.     

Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.     

Actin filament capping protein from bovine brain.

An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F-actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the 'barbed' end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+-independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker

Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.     

Cofilin-linked changes in actin filament flexibility promote severing

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.     

The variable twist of actin and its modulation by actin-binding proteins

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.     

Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.     

Two of the three actin-binding domains of gelsolin bind to the same subdomain of actin. Implications of capping and severing mechanisms.

Gelsolin binds two monomers in the nucleating complex with G-actin in calcium and caps actin filaments. However, 3 actin-binding domains have been identified within its 6 repeating sequence segments corresponding to S1 S2-3 and S4-6. S1 and S4-6 bind only G-actin whereas S2-3 binds specifically to F-actin. Two of the three domains (S2-3 and S4-6) are required for nucleation and a different pair (S1 and S2-3) for severing. Here we show for the first time that the domains unique to nucleation (S4-6) or severing (S1) compete for the same region on subdomain 1 of G-actin. We further show that S2-3 binds actin monomers weakly in G-buffer conditions and that this interaction persists when S1 or S4-6 are also bound. Thus gelsolin associates with two distinct regions on actin. Since S2-3 does not bind monomeric actin in F-buffer, we suggest that its high affinity 1:1 stoichiometry for filament subunits reflects interaction with two adjacent subunits.     

Human spire interacts with the barbed end of the actin filament

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell.     

Caldesmon inhibits the actin-myosin interaction by changing its spatial orientation and mobility during the ATPase activity cycle

Orientation and mobility of acrylodan fluorescent probe specifically bound to caldesmon Cys580 incorporated into muscle ghost fibers decorated with myosin S1 and containing tropomyosin was studied in the presence or absence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. Modeling of various intermediate states of actomyosin has shown discrete changes in orientation and mobility of the dye dipoles which is the evidence for multistep changes in the structural changes of caldesmon during the ATPase hydrolysis cycle. It is suggested that S1 interaction with actin results in nucleotide-dependent displacement of the C-terminal part of caldesmon molecule and changes in its mobility. Thus inhibition of the actomyosin ATPase activity may be due to changes in caldesmon position on the thin filament and its interaction with actin. Our new findings described in the present paper as well as those published recently elsewhere might conciliate the two existing models of molecular mechanism of inhibition of the actomyosin ATPase by caldesmon.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.