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.     

Formation of actin dimers as studied by small angle neutron scattering

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.     

Control of actin filament length by phosphorylation of fragmin-actin complex

Fragmin is a Ca2(+)-sensitive F-actin-severing protein purified from a slime mold, Physarum polycephalum (Hasegawa, T., S. Takahashi, H. Hayashi, and S. Hatano. 1980. Biochemistry. 19:2677-2683). It binds to G-actin to form a 1:1 fragmin/actin complex in the presence of micromolar free Ca2+. The complex nucleates actin polymerization and caps the barbed end of the short F-actin (Sugino, H., and S. Hatano. 1982. Cell Motil. 2:457-470). Subsequent removal of Ca2+, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca2+ concentration. Here we report that this activity of fragmin-actin complex can be abolished by phosphorylation of actin of the complex. When crude extract from Physarum plasmodium was incubated with 5 mM ATP and 1 mM EGTA, the activities of the complex decreased to a great extent. The inactivation of the complex in the crude extract was not observed in the presence of Ca2+. In addition, the activities of the complex inactivated in the crude extract were restored under conditions suitable for phosphatase reactions. We purified factors that inactivated fragmin-actin complex from the crude extract. These factors phosphorylated actin of the complex, and the activities of the complex decreased with an increased level of phosphorylation of the complex. These factors, termed actin kinase, also inactivated the complex that capped the barbed end of short F-actin, leading to elongation of the short F-actin to long F-actin. Thus the length of F-actin can be controlled by phosphorylation of fragmin-actin complex by actin kinase.     

Ca2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization

Gelsolin complexes with calcium (gelsolin-Ca2+) binds to the ends of actin filaments to which monomers add preferentially during elongation. It forms a stable complex with actin in a low ionic strength solution which does not normally favor the polymerization of actin. Gelsolin-Ca2+ increases the rate of nucleation of actin which precedes polymerization, but decreases the rate of elongation of the filaments. The final average length of filaments formed in the presence of gelsolin-Ca2+ is shorter and the equilibrium monomer concentration increases relative to actin polymerized in the absence of gelsolin-Ca2+. Gelsolin-Ca2+ also increases the number of actin filaments because the magnitude of the increase in monomer concentration is disproportionately small compared with the reduction in polymer length. In these respects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of actin filaments is the primary mechanism for the dissolution of an actin gel by gelsolin. Therefore, the ability of gelsolin to produce short filaments irrespective of the initial state of assembly of the actin offers flexibility for controlling the network structure of the cytoplasm in which either the monomeric or polymeric form of actin molecules might predominate at different times.     

Effect of the length and effective diameter of F-actin on the filament orientation in liquid crystalline sols measured by x-ray fiber diffraction.

We examined factors that affect the filament orientation in F-actin sols to prepare highly well-oriented liquid crystalline sols suitable for x-ray fiber diffraction structure analysis. Filamentous particles such as F-actin spontaneously align with one another when concentrated above a certain threshold concentration. This alignment is attributed to the excluded volume effect of the particles. In trying to improve the orientation of F-actin sols, we focused on the excluded volume to see how it affects the alignment. The achievable orientation was sensitive to the ionic strength of the solvent; the filaments were better oriented at lower ionic strengths, where the effective diameter of the filament is relatively large. Sols of longer filaments were better oriented than those of shorter filaments at the same concentration, but the best achievable orientation was limited, probably because of the filament flexibility. The best strategy for making well-oriented F-actin sols is therefore to concentrate F-actin filaments of relatively short length (<1 micrometer) by slow centrifugation in a low-ionic-strength solvent (<30 mM).     

Orientation dependence of displacements by a single one-headed myosin relative to the actin filament.

Displacements of single one-headed myosin molecules in a sparse myosin-rod cofilament were measured from bead displacements at various angles relative to an actin filament by dual optical trapping nanometry. The sparse myosin-rod cofilaments, 5-8 micron long, were synthesized by slowly mixing one-headed myosin prepared by papain digestion with myosin rods at molar ratios of 1:400 to 1:1500, so that one to four one-headed myosin molecules were on average scattered along the cofilament. The bead displacement was approximately 10 nm at low loads ( approximately 0.5 pN) and at angles of 5-10 degrees between the actin and myosin filaments (near physiologically correct orientation). The bead displacement decreased with an increase in the angle. The bead displacement at nearly 90 degrees was approximately 0 nm. When the angle was increased to approximately 150 degrees-170 degrees, the bead displacements increased to 5 nm. A native two-headed myosin showed similar size and orientation dependence of bead displacements as a one-headed myosin.     

Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.     

Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry

Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To date, the resolving ability of 2-DE for protein separation is unsurpassed, thus ensuring its use as the fundamental separation method for proteomics. When immobilized pH gradients (IPGs) are used for isoelectric focusing in the first dimension, excellent reproducibility and high protein load capacity can be achieved. While this has been beneficial for separations of soluble and mildly hydrophobic proteins, separations of membrane proteins and other hydrophobic proteins with IPGs have often been poor. Stimulated by the growing interest in proteomics, recent developments in 2-DE methodology have been aimed at rectifying this situation. Improvements have been made in the area of protein solubilization and sample fractionation, leading to a revamp of traditional approaches for 2-DE of membrane proteins. This review explores these developments.     

Temperature dependence of filament length of Anabaena spiroides Klebahn var. crassa Lemm.

Temperature dependence of filament length ofAnabaena spiroides Klebahn var.crassa Lemm. was examined for a strain isolated from Lake Kasumigaura, Japan. The length of the algal filaments is shown to have good correspondence with the thermal master reaction of cell multiplication.     

The effect of branching on the critical concentration and average filament length of actin

The dependences of the steady-state critical concentration and average filament length of actin solutions, on the filament branching and capping rates, are calculated using a rate methodology based on the total number of actin filaments. The methodology generalizes calculations of the "treadmilling" actin concentration at which an average filament has net zero growth rate. The predictions of the rate methodology are validated by comparison with stochastic-growth simulations that track the positions of all filament subunits over time. For side branching, the critical concentration drops proportionally to the square root of the branching rate; for end branching the drop is linear. The polymerization response to branching has a maximum as a function of the capping-protein concentration. The average filament length drops with increasing branching, because the critical concentration drops. Even small rates of filament uncapping have a large impact on the average filament length in vitro. The potential significance of these phenomena for cell behavior is evaluated.     

A theoretical analysis of filament length fluctuations in actin and other polymers

Control of the structure and dynamics of the actin cytoskeleton is essential for cell motility and for maintaining the structural integrity of cells. Central to understanding the control of these features is an understanding of the dynamics of actin filaments, first as isolated filaments, then as integrated networks, and finally as networks containing higher-order structures such as bundles, stress fibers and acto-myosin complexes. It is known experimentally that single filaments can exhibit large fluctuations, but a detailed understanding of the transient dynamics involved is still lacking. Here we first study stochastic models of a general system involving two-monomer types that can be analyzed completely, and then we report stochastic simulations on the complete actin model with three monomer types. We systematically examine the transient behavior of filament length dynamics so as to gain a better understanding of the time scales involved in reaching a steady state. We predict the lifetime of a cap of one monomer type and obtain the mean and variance of the survival time of a cap at the filament end, which together determine the filament length fluctuations.     

The optical birefringence of DNA solutions induced by oscillatory electric and hydrodynamic fields

The optical birefringence induced in DNA solutions by both oscillating hydrodynamic fields (flow birefringence) and oscillating electric fields (Kerr effect) is measured over a wide frequency range. The observed frequency response of the birefrigence is compared with theories for rigid ellipsoidal particles and for Gaussian chains. DNA at 6 × 10⁵ molecular weight is found to exhibit rigid particle hydrodynamic behavior, while DNA at 5 × 10⁶ molecular weight behaves like a flexible chain. Characterization of the hydrodynamic relaxation spectra for the DNA's by oscillatory flow birefringence allows precise comparison between theory and the experimental Kerr effect response. The dielectric model for DNA contains both permanent and dispersionless induced dipole moments. The dielectric behavior of DNA has the character of a permanent dipole but with anomalous low-frequency dispersion in the Kerr effect. The existing theories do not adequately describe this dispersion. A fluctuation dipole mechanism with relaxation times comparable to those associated with the hydrodynamic motion could possibly demonstrate the observed polar behavior.     

Smooth muscle length adaptation and actin filament length: a network model of the cytoskeletal dysregulation

Length adaptation of the airway smooth muscle cell is attributable to cytoskeletal remodeling. It has been proposed that dysregulated actin filaments may become longer in asthma, and that such elongation would prevent a parallel-to-series transition of contractile units, thus precluding the well-known beneficial effects of deep inspirations and tidal breathing. To test the potential effect that actin filament elongation could have in overall muscle mechanics, we present an extremely simple model. The cytoskeleton is represented as a 2-D network of links (contractile filaments) connecting nodes (adhesion plaques). Such a network evolves in discrete time steps by forming and dissolving links in a stochastic fashion. Links are formed by idealized contractile units whose properties are either those from normal or elongated actin filaments. Oscillations were then imposed on the network to evaluate both the effects of breathing and length adaptation. In response to length oscillation, a network with longer actin filaments showed smaller decreases of force, smaller increases in compliance, and higher shortening velocities. Taken together, these changes correspond to a network that is refractory to the effects of breathing and therefore approximates an asthmatic scenario. Thus, an extremely simple model seems to capture some relatively complex mechanics of airway smooth muscle, supporting the idea that dysregulation of actin filament length may contribute to excessive airway narrowing.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Thermodynamics and kinetics of actin filament nucleation.

We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.     

Effects of chlorpromazine on actin polymerization: slackening of filament elongation and filament annealing.

We have analyzed the effect of chlorpromazine (CPZ) on pure actin. We have found that CPZ quenches Trp-79 and Trp-86 fluorescence and, in agreement with an earlier report on conventional actin, inhibits actin polymerization, lowering the extent of polymerization. Moreover, novel polymerization data are presented indicating that CPZ decreases the maximum polymerization rate in a dose-dependent manner. The assembly inhibition results from the slackening of oligomer formation during the early stages of polymerisation, of filament elongation and of filament annealing. Finally, CPZ strongly inhibits actin filament network formation.     

Change of handedness in cholesteric liquid crystalline phase for N-phthaloylchitosan solutions in organic solvents

The influence of concentration on the helicoidal change of N-phthaloylchitosan (PhCh) solutions in Me2SO, DMAc and DMF was investigated by means of circular dichroism (CD). The critical concentrations to form liquid crystal phase in these three solvents were 43, 45 and 48 wt.%, respectively as measured with polarized optical microscope. There were two kinds of CD peaks, sharp peaks with absorption maximum at about 330 nm induced by the helical conformation of molecular chain, and very broad peaks covering almost whole visible region induced by the cholesteric helix of mesophase. The later only appeared in concentrated solutions with the concentration higher than the critical concentration. The handedness of both levels of helicoidal structures changed from left- to right-handed with the increase of concentration for PhCh/Me2SO solutions. The chirality transfer occurred between these two chiral levels. For PhCh/DMAc and PhCh/DMF systems, only the handedness of helical conformation reversed, but the cholesteric helix did not change. As a method to measure critical concentration, CD is more sensitive than polarized optical microscopy (POM).     

Head group and chain length dependence of phospholipid self-assembly studied by spin-label electron spin resonance

The critical micelle concentrations (cmc's) of a variety of spin-labeled phospholipids, 1-acyl-2-[4-(4,4-dimethyloxazolidine-N-oxyl)valeryl]-sn-glycero-3-pho sph o derivatives, have been determined by electron spin resonance (ESR) spectroscopy. The narrow, three-line ESR spectra of the rapidly tumbling monomers are clearly distinguished from the spin-spin broadened spectra of the micellar aggregates, allowing a direct determination of the concentrations of the two species. The influence of both the hydrocarbon chain length and the polar head group on the energetics of self-assembly has been studied. For phosphatidylcholine, 1n [cmc] decreases linearly with the length of the sn-1 chain. The gradient of this linear dependence corresponds to a free energy of transfer of the monomer from the aqueous phase to the micelle of delta Gtr = -1.1RT per CH2 group. The cmc's of the 1-lauroyl derivatives of both phosphatidylcholine and phosphatidylglycerol have relatively shallow, biphasic temperature dependences with a minimum at approximately 20 degrees C. Both of these properties are characteristic of the hydrophobic effect, with the free energy of transfer being slightly less than that for the solubility of n-hydrocarbons in water, corresponding to the reduced configurational entropy of the lipid chains in the micellar state. The cmc's of the 1-lauroyl derivatives of the phospholipids in 0.15 M NaCl, for their various charge states, are as follows: phosphatidic acid(2-), 0.77 mM; phosphatidic acid(1-), 0.13 mM; phosphatidylserine(1-), 0.24 mM; phosphatidylglycerol(1-), 0.17 mM; phosphatidylcholine, 0.10 mM; phosphatidylethanolamine, 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)