Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.     

Cross-helix separation of tropomyosin molecules in acto-tropomyosin as determined by neutron scattering.

The cross-helix separation of Tm molecules in acto-tropomyosin has been determined using neutron scattering. Deuterated Dictyostelium discoideum actin was density matched in a 93% D2O buffer so that effectively only the protonated tropomyosin was "visible" to neutrons. Analysis of the solution scattering pattern in the region of the first oscillation yielded a value for the cross-helix separation of 7.9 +/- 0.3 nm. The implications of this value for the mechanism of the regulation of muscle contraction are discussed in light of recent results by others.     

Mechanism of force generation studied by neutron scattering

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered “invisible” to neutrons when dissolved in 94% D₂O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometrics, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.     

Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.     

A nonlinear mathematical model of DNA and its use in calculating neutron scatter

A simple nonlinear mathematical model is applied to calculate spectrum of inelastic coherent scattering of neutrons by DNA.     

Insight into the kinetics and the mode of the interaction between smooth muscle calponin and F-actin

Kinetics of the smooth muscle calponin-F-actin interaction was studied by stopped-flow measurements of light scattering and fluorescence intensity of pyrene-labelled F-actin. The intensity and character of the changes in light scattering, and thus the mode of calponin binding to actin filaments leading to changes in their shape and bundling, depend on the molar ratio of the two proteins. Parallel measurements of pyrene-fluorescence quenching upon calponin binding revealed that intrinsic conformational changes in actin filaments are delayed relative to the binding process and are not markedly influenced by the mode of calponin binding. Bundling of actin filaments by calponin was not correlated with fluorescence changes and thus with alterations in the structure of actin filaments.     

Time-resolved X-ray scattering study of actin polymerization from profilactin

The polymerization of actin in solutions of purified calf spleen actin or profilactin (1–10 mg·ml^-1) was followed by synchrotron radiation X-ray solution scattering. At the concentration used, polymerization of actin from profilactin or actin occurs without any lag phase. It is shown by a combination of solution scattering, model calculations and electron microscopy that contrary to the conclusions from previous viscometry studies, filaments form without any lag phase in profilactin solution but aggregate in bundles or networks. This phenomenon is independent of the method used to induce polymerization: slow temperature increase, temperature jump in the presence of polymerizing salts or fast mixing with salt. This aggregation explains the lower final viscosity levels, as compared to actin solutions, observed during the polymerization of actin from profilactin.     

Properties of the interaction between phosphofructokinase and actin

The interaction of rabbit skeletal muscle phosphofructokinase (PFK) with actin is characterized in terms of the binding of PFK to actin in the presence and absence of tropomyosin and troponin, the effect of PFK on actin polymerization, and the involvement of adenylates in the binding of PFK to actin. The thin filament proteins, tropomyosin and troponin, are associated with skeletal muscle actin and reduce the binding of PFK to actin, thus influencing the probable distribution of PFK in skeletal muscle. The binding of PFK to actin is inhibited by ATP and ADP but not by fructose 6-phosphate or fructose 2,6-bisphosphate. This specific inhibition, plus evidence from fluorescence quenching and photoaffinity labeling, suggests that actin binds at the adenosine activation sites of PFK. Light scattering measurements used to monitor actin polymerization indicate that PFK dramatically increases the level of light scattering produced by the polymerization of actin, indicative of a superaggregate of PFK and actin. PFK inhibits the polymerization of actin when polymerization is induced by low concentrations of added salts. Although PFK binds to actin with high affinity, it seems to have little effect on the high shear viscosity of actin filaments.     

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.     

The effect of organic cryosolvents on actin structure: studies by small angle X-ray scattering

Small-angle X-ray scattering was used to probe the structure of actin in the presence of cryosolvents: 1,2-propanediol, glycerol, or a mixture of both solvents. In media devoid of polymerizing salts, a radius of gyration of 23 Å is measured, as expected from the literature. In the presence of 1,2-propanediol alone, the scattering pattern begins to exhibit the characteristic slope of elongated objects with a non-negligible thickness, such as actin filaments polymerized in 40 mM KCl and 1 mM MgCl₂. However, only short fragments (radius of gyration 40 Å) are generated. We infer that in a medium of low ionic strength containing 15% 1,2-propanediol, actin assumes a structure closer to that of filamentous actin. 1,2-propanediol apparently induces nucleation of oligomers, as with polymerizing salts, but no propagation occurs. Glycerol and/or propanediol induce no alteration in the structure of individual salt-polymerized actin filaments. Aggregation occurs with propanediol, even in the presence of glycerol. Glycerol alone has no such effect. No shortening is detected within the scale covered, with either solvent, although 1,2-propanediol is known to shorten actin filaments. We suggest that in the absence of salts, 1,2-propanediol induces a conformational change in monomeric actin that is necessary for nucleation. This could correlate with a conformational change of actin protomers within microfilaments observed in the presence of 1,2-propanediol by other authors using different techniques.Abbreviations SAXS small-angle X-ray scattering - G-actin globular monomeric actin - F-actin filamentous polymerized actin Correspondence to: E. Pajot-Augy     

Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.     

Skeletal muscle myosin subfragment-1 induces bundle formation by actin filaments

As is well known, the light scattering intensity of F-actin solutions increases immediately upon formation of the rigor complex with subfragment-1 (S-1). We have found that after the initial rise in scattering, there is a further gradual increase in scattering (we call it "super-opalescence"). Fluorescence and electron microscopic observations of acto-S-1 solutions showed that super-opalescence results from formation of actin filament bundles once S-1 binds to F-actin. The actin bundles possessed transverse stripes with a periodicity of about 350 A, which suggested that in the bundles actin filaments are arranged in parallel register. The rate of the initial process of bundle formation (i.e. side-by-side dimerization) could be approximately estimated by measuring the initial rate of super-opalescence (V0). V0 had a maximum (V0m) at a molar ratio of S-1 to actin of 1;6-1;7, regardless of the actin concentration, pH (6-8.5), Mg2+ concentration (up to 5 mM), or ionic strength (up to 0.3 M KC1). Lower pH, higher Mg2+ concentration, and higher ionic strength increased V0m; V0 was proportional to the square of the actin concentration, regardless of the solution conditions.     

Conformational changes of troponin C within the thin filaments detected by neutron scattering

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered "invisible" to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 A (99% confidence limits between 22 A and 23 A) and 24 A (99% confidence limits between 23 A and 25 A) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 A (99% confidence limits between 49 A and 57 A) and 49 A (99% confidence limits between 44 A and 53 A) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4A is associated with large conformational changes of the entire Tn molecule by binding of Ca2+.     

Severing of F-actin by yeast cofilin is pH-independent

Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.     

Comparison of neutron and X-ray scattering of dilute myoglobin solutions.

Experimental results obtained by neutron scattering of dilute solutions of myoglobin are compared with those obtained by X-ray scattering. X-ray scattering remains the more powerful technique at wider angles above 0.3 Å⁻¹, where neutron experiments are less accurate because of low coherent scattering probability and high incoherent background. Neutron scattering is preferable at momentum transfers below 0.2 Å⁻¹; the conditions for applying the contrast variation method for the evaluation of the three basic scattering functions, which are due to shape and internal structure, equation (3), are ideally fulfilled in this region. Furthermore, neutrons allow observation of the hydrogen-deuterium exchange within the protein.     

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.     

Synapsin IIa Bundles Actin Filaments

Abstract: Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.     

Structure of myosin subfragment 1 from low-angle X-ray scattering

The X-ray scattering pattern produced by a solution of myosin subfragment 1 has been measured to a resolution (Bragg spacing) of 2 nm. We find that for subfragment 1 (S1) prepared by limited papain digestion in the presence of ethylenediaminetetraacetate the radius of gyration is 3.28 +/- 0.06 nm, the volume is 151 +/- 6 nm3, the surface area is 330 +/- 15 nm2, and the length of the maximum chord is 12.0 +/- 1.0 nm. The theoretical scattering patterns from several objects of uniform electron density have been calculated and compared with the observed scattering produced by S1. The recent three-dimensional electron micrograph reconstruction of S1-decorated actin by J. Seymour and E. O'Brien (private communication) generated the calculated pattern that best fit the observed scattering. This fit strongly suggests that this reconstruction resembles subfragment 1. The good correspondence between an S1 structure derived when S1 is attached to actin and a study of free S1 in solution strongly suggests that binding to actin does not grossly distort the shape of S1. This is consistent with the notion that S1 changes its orientation on actin, rather than its shape, in order to generate the contractile force in muscle.     

Dynamical Form-Factor of Neutron Scattering by DNA Solitons

Method of scattering of slow neutrons has been considered in recent years as a rather perspective way to study the nonlinear DNA dynamics. In this paper we present the results of theoretical calculations of the dynamical form-factor of the scattering. The calculations were made on the basis of the nonlinear mathematical model which takes into account rotational motions of bases around the sugar-phosphate chains. The results of the calculations are considered as predictions for further neutron scattering experiments.     

Rate and mechanism of the assembly of tropomyosin with actin filaments

The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM MgCl2, 100 mM KCl, pH 7.5, 25 degrees C) and at the protein concentrations used (2.5-24 microM actin, 0.2-3.4 microM tropomyosin) the half-life time of assembly of tropomyosin with actin filaments was found to be less than 1 s. The results were analyzed quantitatively by a model in which tropomyosin initially binds to isolated sites. Further tropomyosin molecules bind contiguously to bound tropomyosin along the actin filaments. Good agreement between the experimental and theoretical time course of assembly was obtained by assuming a fast preequilibrium between free and isolatedly bound tropomyosin.