Probe diffusion in solutions of filamentous actin formed in the presence of gelsolin.

Dynamic light scattering was used to characterize the diffusion of monodisperse polystyrene latex spheres (PLS) of different sizes (55-, 105-, and 265-nm radii) in column-purified 0.65 mg/mL actin solutions polymerized with 100 mM KCl in the absence and presence of various amounts of the actin-binding protein gelsolin. The gelsolin and its interaction with actin was initially studied to ensure that the gelsolin could be used to produce filament populations with well-defined mean lengths. Measurements with PLS diffusion probes present showed, in the absence of gelsolin, that the effective local microviscosity in the actin solutions was 5-20 times that of water and that a large fraction of the PLS are trapped within the pores of the actin filament network, as found previously [J. Newman, K. L. Schick, & K. S. Zaner, (1989) Biopolymers 28, 1969-1980]. As the molar ratio of gelsolin to actin was increased, the diffusion coefficients of the PLS approached those in pure water while the degree of PLS trapping went to zero. Measurements of the dependence of the PLS diffusion coefficients on the ratio of actin concentration to the semidilute overlap concentration showed, for the smaller PLS, a transition occurring near the mean global overlap concentration. These results reflect the dissolution of the actin network as the gelsolin concentration is increased and illustrate the role of gelsolin/actin interactions in the control of macromolecular transport within the periphery of cells.     

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.     

Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus

1. The kinetics of decay in the dark of the transmembrane pH difference (delta pH) induced by light in nonphosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9-aminoacridine, in the presence of 50 mM KCl and 2 microM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-228]. The kinetic competence of the probe response was tested by accelerating the delta pH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different delta pH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 micron.s-1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of delta pH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K(+)-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determinants of the translational mobility of a small solute in cell cytoplasm

The purposes of this study were: (a) to measure the translational mobility of a small solute in cell cytoplasm; (b) to define quantitatively the factors that determine solute translation; and (c) to compare and contrast solute rotation and translation. A small fluorescent probe, 2,7-bis-(2-carboxyethyl)-5-(and 6-)- carboxyfluorescein (BCECF), was introduced into the cytoplasm of Swiss 3T3 fibroblasts. BCECF translation was measured by fluorescence recovery after photo-bleaching; rotation was measured by Fourier transform polarization microscopy. Diffusion coefficients relative to those in water (D/D0) were determined by comparing mobility in cytoplasm with mobility in standard solutions of known viscosity. At isosmotic cell volume, the relative diffusion coefficients for BCECF translation and rotation in cytoplasm were 0.27 +/- 0.01 (SEM, n = 24, 23 degrees C) and 0.78 +/- 0.03 (n = 4), respectively. As cell volume increased from 0.33 to 2 times isosmotic volume, the relative translational diffusion coefficient increased from 0.047 to 0.32, while the relative rotational diffusion coefficient remained constant. The factors determining BCECF translation were evaluated by comparing rotation and translation in cytoplasm, and in artificial solutions containing dextrans (mobile barriers) and agarose gels (immobile barriers). It was concluded that the hindrance of BCECF translation in cytoplasm could be quantitatively attributed to three independent factors: (a) fluid-phase cytoplasmic viscosity is 28% greater than the viscosity of water (factor 1 = 0.78); (b) 19% of BCECF is transiently bound to intracellular components of low mobility (factor 2 = 0.81); and most importantly, (c) translation of unbound BCECF is hindered 2.5- fold by collisions with cell solids comprising 13% of isosmotic cell volume (factor 3 = 0.40). The product of the 3 factors is 0.25 +/- 0.03, in good agreement with the measured D/D0 of 0.27 +/- 0.01. These results provide the first measurement of the translational mobility of a small solute in cell cytoplasm and define quantitatively the factors that slow solute translation.     

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).     

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     

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.     

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.     

F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid crystalline solutions.

Single actin filaments were analyzed in solutions ranging from dilute (0.2 microgram/ml), where filaments interact only with solvent, to concentrations (4.0 mg/ml) at which F-actin forms a nematic phase. A persistence length of approximately 1.8 microns and an average length of approximately 22 microns (Kaufmann et al., 1992) identify actin as a model for studying the dynamics of semiflexible polymers. In dilute solutions the filaments exhibit thermal bending undulations in addition to diffusive motion. At higher semidilute concentrations (1.4 mg/ml) three-dimensional reconstructions of confocal images of fluorescently labeled filaments in a matrix of unlabeled F-actin reveal steric interactions between filaments, which account for the viscoelastic behavior of these solutions. The restricted undulations of these labeled chains reveal the virtual tube formed around a filament by the surrounding actin. The average tube diameter <a> scales with monomer concentration c as <a> varies; is directly proportional to c-(0.5 +/- 0.15). The diffusion of filaments in semidilute solutions (c = (0.1-2.0) mg/ml) is dominated by diffusion along the filament contour (reptation), and constraint release by remodeling of the surrounding filaments is rare. The self-diffusion coefficient D parallel along the tube decreases linearly with the chain length for semidilute solutions. For concentrations > 2.5 mg/ml a transition occurs from an isotropic entangled phase to a coexistence between isotropic and nematic domains. Analysis of the molecular motions of filaments suggests that the filaments in the aligned domains are in thermal equilibrium and that the diffusion coefficient parallel to the director D parallel is nearly independent of filament length. We also report the novel direct observation of u-shaped defects, called hairpins, in the nematic domains.     

Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

The bundling of actin with polyethylene glycol 8000 in the presence and absence of gelsolin.

Actin filament and bundle formation occur in the cytosol under conditions of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a means of simulating crowded conditions in vitro. Column-purified Ca-actin was polymerized in the absence and presence of gelsolin (to regulate mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCl and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by fluorescence and phase-contrast microscopy. The minimum concentration of KCl required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cation, bundling is reversible on dilution, as determined by intensity levels, diffusion coefficients, and microscopy. However, with either 2 mM Mg2+ or Ca2+ added, bundling is irreversible under conditions of higher PEG concentrations or longer filaments, indicating that osmotic pressure effects cannot fully explain actin bundling with PEG. Weaker divalent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.     

Correlation between biological activity of 30 S subunits of Escherichia coli ribosomes and their conformation changes revealed by optical mixing spectroscopy

Spectral analysis of light scattered from solutions of 30 S subunits was performed by the method of regularization of the inverse spectral problem. The subunits observed under ionic conditions which preserved their biological activity (200 mM NH4Cl at 1 mM MgCl2) revealed a monodisperse pattern of scattering with diffusion constant D = (1.83 +/- 0.10) X 10(-7) cm2/s. The polydispersity and compaction of 30 S subunits were observed under inactivation ionic conditions (30 mM NH4Cl at 1 mM MgCl2). The number of compacted particles correlates with the irreversible loss of biological activity, the ability of 30 S subunits to bind specific tRNA.     

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

Structure and dynamics of M13mp19 circular single-strand DNA: effects of ionic strength

Dynamic and static light scattering, CD, and optical melting experiments have been conducted on M13mp19 viral circular single-strand DNA as a function of NaCl concentration. Over the 10,000-fold range in concentration from 100 microM to 1.0 M NaCl, the melting curves and CD spectra indicate an increase in base stacking and stability of stacked regions with increased salt concentration. Analysis of dynamic light scattering measurements of the single-strand DNA solutions as a function of K2 from 1.56 to 20 X 10(10) cm-2 indicates the collected autocorrelation functions are biexponential, thus revealing the presence of two decaying dynamic components. These components are taken to correspond to (1) global translational motions of the molecular center of mass and (2) motions of the internal molecular subunits. From the evaluated relaxation rates of these components, diffusion coefficients D0 and Dplat are determined. The center of mass translational diffusion coefficient D0, varies in a nonmonotonic manner, by 10%, from 3.75 X 10(-8) to 3.39 X 10(-8) cm2/s over the NaCl concentration range from 100 microM to 1.0 M. Likewise, the radius of gyration RG, obtained from static light scattering experiments, varies by 15% from 699 to 830 A over the same NaCl range Dplat, the diffusion coefficient of the internal subunits, displays a different dependence on the NaCl concentration and decreases, by nearly 22% in a titratable fashion, from 12.46 X 10(-8) to 10.26 X 10(-8) cm2/s, when the salt is increased from 100 microM to 1.0 M. A semiquantitative interpretation of these results is provided by analysis of the light scattering data in terms of the circular Rouse-Zimm chain. Rouse-Zimm model parameters are estimated from the experimental results, assuming the circular chains are composed of a fixed number of Gaussian segments, N + 1 = 15. The rms displacement of the internal segments, b, is estimated to be the smallest (442 A) in 100 mM NaCl. Increases of b to 467 A in 100 microM and 524 A in 1.0 M NaCl are observed. Meanwhile, the hypothetical friction factor of the internal subunits, f, progressively increases as the NaCl concentration is raised. It is inferred from the evaluated Rouse-Zimm model parameters that both the static flexibility of the circular chain and diffusive displacements of the internal subunits decrease with increases in NaCl concentration from 100 mM to 1.0 M.(ABSTRACT TRUNCATED AT 400 WORDS)     

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast- twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half- width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).     

Characterization of oligomers as kinetic intermediates in myosin subfragment 1-induced polymerization of G-actin.

The nature of the kinetic intermediates involved in S1-induced polymerization of G-actin into F-acto-S1-decorated filaments has been investigated using as probes light scattering, the fluorescence of pyrenyl- or NBD-labeled actin, and the anisotropy of fluorescence of N-iodoacetyl-N'-(5-sulfo-1-napthyl)ethylene diamine (AEDANS)-labeled actin. With AEDANS-G-actin, the initial formation of a ternary G2S complex between two G-actin and one S1 molecules (Valentin-Ranc, C., Combeau, C., Carlier, M. F., and Pantaloni, D. (1991) J. Biol. Chem. 266, 17871-17879) has been confirmed. Data obtained with all probes are consistent with the subsequent rapid formation of G-actin-S1 oligomers in which the actin/S1 molar ratio is 2:1. Oligomers form above 0.6 microM G-actin with S1(A1) and above 3.5 microM G-actin with S1(A2), at 20 degrees C. Oligomerization is endothermic with a delta H of 14 kcal/mol. A tentative model is proposed to comprehensively account for the data and the structural features of the F-actin-S1 filament. Within this model, longitudinal actin-actin interactions take place in G2S, and lateral, hydrophobic actin-actin interactions appear upon formation of (G2S)n oligomers.     

Acanthamoeba profilin binding to fluorescein-labeled actins.

The binding constants of Acanthamoeba profilin to fluorescein-labeled actin from Acanthamoeba and from rabbit skeletal muscle have been determined by measuring the reduction in the actin tracer diffusion coefficients, determined by fluorescence photobleaching recovery, as a function of added profilin concentration. Data were analyzed using a two-parameter nonlinear regression analysis to determine the profilin-actin dissociation constant Kd and the profilactin diffusion coefficient, DPA. For fluorescein-labeled Acanthamoeba actin, the least-squares estimates for Kd and DPA, along with approximate single standard deviation confidence intervals, are Kd = 48 (36, 63) microM and DPA = 6.72 (6.62, 6.81) X 10(-7) cm2s-1. For fluorescein-labeled skeletal muscle actin, the corresponding values are Kd = 147 (94, 225) microM and DPA = 6.7 (6.3, 7.0) X 10(-7) cm2s-1. These dissociation constants are the first to be determined from direct physical measurement; they are in agreement with values inferred from earlier studies on the effect of profilin on the assembly of actin that had been fluorescently labeled or otherwise modified at Cys 374. These results place important restrictions on the interpretation of experiments in which fluorescently labeled actin is used as a probe of living cytoplasm or cytoplasmic extracts that include profilin.     

Quasielastic light scattering study of thermal excitations of F-actin solutions and of growth kinetics of actin filaments.

In the first part of this work we report quasielastic light scattering (QELS) studies of the internal dynamics of transient actin networks over a time range of 10(-6)-10(-2) s, scattering angles between zeta = 20 degrees and 150 degrees, and a concentration range of 0.015 (0.3) to 0.7 mg/mL (15 microM). We confirm our previous result that (1) the dynamic structure factor g(q,t) is determined by the thermally excited undulations of the actin filaments and (2) that the initial decay of g(q, t) scales as g(q, t) varies; is directly proportional to exp(-q alpha t) while the long time decay scales as g(q, t) varies; is directly proportional to exp [-(Aq alpha t) 2/3] with alpha = 2.75. The deviation of alpha from the theoretical value of alpha = 3 predicted for Rouse-Zimm chains is similar to that found for high molecular weight macromolecular solutions by QELS. A refined analysis of the dynamic structure factor showed that it can be interpreted in terms of three relaxation processes (besides the contribution of the residual monomer diffusion): (1) the dominant Rouse-Zimm dynamics, which comprises between 65 (at high concentrations) and 85% of the signal; (2) a fast relaxation process with a decay constant of gamma = 9 x 10(3) s-1, which contributes at all concentrations with the same amplitude; and (3) a nonexponential ultraslow contribution of the form g(us) varies; is directly proportional to exp [(-gamma ust)]1/4. The third contribution appears only at high concentrations and increases strongly with decreasing scattering angles. It is thus attributed to fluctuations of the mesh size of the transient actin network. In the second part we show that high sensitivity QELS may be applied to follow the actin polymerization process at low temperatures (10 degrees C). The apparent diffusion coefficient and the static scattering intensity of the actin filaments were determined as functions of polymerization time tpol. We show that the process consists of the rapid growth of a few filaments that become very long (approximately 10 microns; even at actin concentrations of 0.04 micrograms/mL) near the critical growth concentration of 0.012 micrograms/mL, as is expected for a growth process determined by nucleation. Finally, we studied actin networks polymerized in the presence of complexes of gelsolin with actin. By application of the CONTIN program we could determine the length distribution of the filaments.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.