Surface-induced polymerization of actin

Living cells contain a very large amount of membrane surface area, which potentially influences the direction, the kinetics, and the localization of biochemical reactions. This paper quantitatively evaluates the possibility that a lipid monolayer can adsorb actin from a nonpolymerizing solution, induce its polymerization, and form a 2D network of individual actin filaments, in conditions that forbid bulk polymerization. G- and F-actin solutions were studied beneath saturated Langmuir monolayers containing phosphatidylcholine (PC, neutral) and stearylamine (SA, a positively charged surfactant) at PC:SA = 3:1 molar ratio. Ellipsometry, tensiometry, shear elastic measurements, electron microscopy, and dark-field light microscopy were used to characterize the adsorption kinetics and the interfacial polymerization of actin. In all cases studied, actin follows a monoexponential reaction-limited adsorption with similar time constants (approximately 10(3) s). At a longer time scale the shear elasticity of the monomeric actin adsorbate increases only in the presence of lipids, to a 2D shear elastic modulus of mu approximately 30 mN/m, indicating the formation of a structure coupled to the monolayer. Electron microscopy shows the formation of a 2D network of actin filaments at the PC:SA surface, and several arguments strongly suggest that this network is indeed causing the observed elasticity. Adsorption of F-actin to PC:SA leads more quickly to a slightly more rigid interface with a modulus of mu approximately 50 mN/m.     

Polyamine-induced actin polymerization.

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain length of the polyamine. Among the biological polyamines tested, spermidine and spermine are the most efficient. The polymerization of actin can also be induced by the corresponding mono or diguanidine derivatives of these polyamines but monoamines or amino acids are inactive at the same concentration. The transformation of actin from a globular to a fibrous from upon addition of spermidine is also demonstrated by the changes in the near-ultraviolet circular dichoroic spectrum of this protein. Moreover, the polyamine-induced F -actin exhibits the same properties as the salt-induced F -actin: it strongly activates the Mg2+ -ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.     

Amplification of actin polymerization forces

The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

Introduction

Mechanical amplification is something we experience every day, in the form of gears, pulleys, and levers. While climbing a hill on a bicycle, for instance, shifting gears increases the force on the wheels while limiting the pressure required on the pedals. However, energy has to be conserved, and because mechanical work is defined as force × displacement, an increase in force can only be obtained at the expense of displacement. Thus, although shifting gears allows one to develop the additional force needed to go uphill, speed is reduced as each pedal stroke produces a smaller turn of the wheels. Cells have similarly developed microscopic force amplification strategies during evolution. Here, we discuss some amplification schemes for one of the major force generators in the cell—actin polymerization.Actin plays a ubiquitous role in cell motility and morphogenesis, spanning many scales of space and time. In fission yeast, for example, a miniature actin machinery only ∼100 nm across can induce the invagination of an endocytic vesicle in just a few seconds (Picco et al., 2015). However, to sever the entire yeast cell, a cytokinetic ring forms with an initial perimeter of ∼10 µm and requires ∼30 min to drive division (Proctor et al., 2012). These assemblies differ dramatically in both size and duration. In other species, considerably larger actin assemblies exist that reach the scale of centimeters, such as in muscle cells. Clearly, actin and its associated factors need to be specifically organized to achieve these different functions (Fig. 1). From a functional point of view, a key problem is to understand how the global architecture of an actin network allows forces that are produced at the molecular scale to be productive for the cell. In this respect, we can distinguish two sorts of components. Active components generate forces from chemical sources of energy and include molecular motors, as well as actin itself, which can push by polymerizing (Kovar and Pollard, 2004) and possibly pull while depolymerizing. Passive components, such as actin cross-linkers, are essential but can only transmit forces generated by other elements.Open in a separate windowFigure 1.Different actin networks. Networks of actin filaments are essential for many biological processes at the cellular level, and the organization of the filaments in space must be adapted to the task. Here, polymerization force (orange) of actin filaments (red) occurs near the plasma membrane (blue). Linear filopodia bundles with fascin (black) can produce high speeds, but represent a weak configuration for force generation. Lamellipodia are thin cellular extensions in which filaments are nearly parallel to the substrate on which the cell is crawling. The 2D branched network, created by Arp2/3 actin-nucleating complexes (black), can produce higher forces at the expense of displacement. During endocytosis in yeast, actin forms a 3D network at the site of the invagination that appears roughly spherical, but the organization of actin filaments in space is not known. The coat structure (yellow) enables actin to pull the membrane inward and actin polymerizes near the base of the structure, where Arp2/3 nucleators are shown in black (Picco et al., 2015). Endocytosis requires strong force amplification to pull the invagination against the turgor pressure.The forces developed by an actin meshwork are determined by the organization of its components. Ultimately, these forces must be sufficient to drive biological processes, and thus their scale depends on the physical characteristics of the cell. For example, in the case of endocytosis in yeast, the turgor pressure pushing the surface of the invagination outward reaches ∼1,000 pN, which the actin machinery must overcome (Basu et al., 2014). During cytokinesis, the actomyosin ring also works against the turgor pressure, which produces high forces on the furrow (Proctor et al., 2012). For both cases, these forces have been calculated from measured cellular parameters, particularly the turgor pressure and the dimensions over which the membrane is deformed. Hence, for these processes at least, the two ends of the problem are known: the forces produced by the molecular components make up the input and the force required for the cellular process to occur represents the output. Yet the force balance within the system must be considered to understand how the actin machinery harvests the input to produce this output.In this comment, we focus on the transmission of forces produced by the polymerization of actin, setting aside turnover and the contribution of molecular motors. We discuss specifically how the arrangement of the filaments in the system regulates the amount of productive force. In many ways, the actin machinery behaves analogously to a cyclist: though its power is limited, it can “shift gears” to favor either more displacement (high gears) or more force (low gears).

The force generated by actin polymerization

Actin polymerization can produce force. Indeed if an actin monomer in solution binds the barbed end of a filament, there is a change of free energy (ΔG_p) and polymerization will occur if ΔG_p < 0 (Fig. 2 A). This reaction depends on the concentration (C) of monomeric actin and will take place only above a critical concentration (C* of ∼0.14 µM; Pollard, 1986). It is associated with ΔG_p = −k_BT ln(C/C*), where k_B is the Boltzmann constant and T is the absolute temperature. If actin is polymerizing against a load and producing work (W), the change in free energy is ΔG_p + W. In this case, polymerization will occur spontaneously if the change is negative, i.e., ΔG_p + W < 0. Consider an actin filament pushing against a force (f) applied parallel to the filament axis (Fig. 2 B). Because the addition of one actin monomer produces a displacement (δ = 2.75 nm; Holmes et al., 1990), the mechanical work is W = f × δ. Forces that are antagonistic to elongation can impede actin assembly (Peskin et al., 1993). The critical force under which the filament would cease to elongate is called the polymerization force (f_a). Using a physiological concentration (C of ∼40 µM; Wu and Pollard, 2005), the polymerization force is thermodynamically limited to k_BT ln(C/C*)/δ = ∼9 pN (Hill, 1981). Within such limits, the force developed by polymerization will depend on the conditions of assembly. Direct measurements of the polymerization force using single-molecule techniques are scarce. A first study used optical traps on bare filaments, giving a force of ∼1 pN (Footer et al., 2007). By monitoring the buckling of filaments capped with formins, a second study found the force to be ∼1.3 pN (Kovar and Pollard, 2004). In both cases, the concentration of actin was an order of magnitude lower than in vivo, and the measured forces were in fact close to the theoretical maximum under the experimental conditions. Here, we will thus consider that f_a is within 1 and 9 pN. We further assume that an actin filament is able to elongate as long as the parallel component of the antagonistic force at its barbed end remains lower than f_a, irrespective of the perpendicular components (Fig. 2 C). We discuss various examples of force amplification in which the network develops forces that exceed f_a per filament, without breaking the thermodynamic requirement for actin polymerization (ΔG_p + W < 0).Open in a separate windowFigure 2.Polymerization mechanics. (A) During polymerization, the addition of one actin monomer (orange) corresponds to an elongation (δ) at the barbed end of an actin filament (red) and is associated with a change of free energy (ΔG_p = −k_b T ln(C/C*)). (B) The work required to push a load over a distance (h) with a force (f) is f × h, and thus assembly remains favorable as long as ΔG_p + f × h < 0. In the case where polymerization occurs straight against a load (h = δ), the maximal force (f_a) is f_a = k_b T ln(C/C*)/δ (Hill, 1981). (C) If the filament encounters the load with an angle (θ), then h = δ sinθ and the maximal force is consequently increased: f_θ = f_a/sinθ. (D) In the branched network of a lamellipod, actin grows against the leading membrane at an angle (θ = ∼54°). In the absence of friction, the force between the polymerizing tip (orange) of the actin and the membrane (blue) is perpendicular to the membrane. It can then reach a maximum magnitude of f_a/sinθ. The sum of the forces produced by the two filaments is then ∼2.5 f_a. (E) Higher forces arise by polymerizing with shallow angles. The device illustrated here is composed of a growing actin filament with a “leg” on its side. By elongating, the filament will induce rotation around the pivot point, where the leg is contacting the membrane. High forces can be exerted on a load supported at the branch point, as a result of the amplification achieved by the lever arm and contact angle. (F) The highest forces are generated if a filament polymerizes parallel to the surface. In the illustrated configuration, elongation of the filament will cause a load (green dome) to separate from the membrane. The maximal force is calculated as in E, except that anchoring has to be assumed at the pivot point to balance forces horizontally. The device can sustain high forces applied on the top of the dome because the upward movement is small compared with the elongation of the filament.

Table 1.

Physical characteristics of actin

Water in actin polymerization.

We have addressed the question whether water is part of the G- to F-actin polymerization reaction. Under osmotic stress, the critical concentration for G-Ca-ATP actin was reduced for six different osmolytes. These results are interpreted as showing that reducing water activity favored the polymerized state. The magnitude of the effect correlated, then saturated, with increasing MW of the osmolyte and suggested that up to 10-12 fewer water molecules were associated with actin when it polymerized. By contrast, osmotic effects were insignificant for Mg-ATP actin. The nucleotide binding site of the Mg conformation is more closed than the Ca and more closely resembles the closed actin conformation in the polymerized state. These results suggest that the water may come from the cleft of the nucleotide binding site.     

Models for actin polymerization motors

Actin polymerization drives cell membrane protrusions and the propulsion of intracellular pathogens. The molecular mechanisms driving actin polymerization are not yet fully understood. Various mathematical models have been proposed to explain how cells convert chemical energy released upon actin polymerization into a pushing force on a surface. These models have attempted to explain puzzling properties of actin-based motility, including persistent attachment of the network to the membrane during propulsion and the interesting trajectories of propelled particles. These models fall generally into two classes: those requiring filament (+)-ends to fluctuate freely from the membrane to add subunits, and those where filaments elongate with their (+)-ends persistently associated with surface through filament end-tracking proteins ("actoclampin" models). This review compares and contrasts the key predictions of these two classes of models with regard to force-velocity profiles, and evaluates them with respect to experiments with biomimetic particles, and the experimental evidence on the role of end-tracking proteins such as formins and nucleation-promoting factors in actin-based motility.     

Isolation and polymerization of brain actin

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar to actin, is the major constituent. A method is described for isolating the “brain actin” to a purity of 90–95%. The isolation method begins with an extraction of bovine brain in low ionic strength buffer with ATP and sucrose. The extract is treated with NH₄SO₄, MgCl, and KCl and incubated at 37°C. A precipitate is formed which contains primarily tubulin and brain actin. Resolubilization of the brain actin is achieved with a low ionic strength buffer solution with sucrose and ATP. Further purification is accomplished by a cycle of polymerization—depolymerization. This “brain actin” shares with muscle actin the following properties: (1) Similar molecular weight and molecular charge as determined by SDS polyacrylamide gel and ordinary disc electrophoresis; (2) Polymerization to a filamentous form under the same conditions; (3) Contains 3-methylhistidine; (4) Vinblastine sulfate will induce filament formation.     

Caldesmon-induced polymerization of actin from profilactin

We have investigated the effect of caldesmon, a Ca2+/calmodulin-regulated actin-binding protein, on the complex between profilin and G-actin (profilactin). We found that smooth muscle caldesmon dissociates this complex rapidly and induces the polymerization of the released actin. Native profilactin (e.g. the complex isolated from calf thymus) proved more resistant to the attack of caldesmon than a heterologous complex reconstituted from calf thymus profilin and skeletal muscle actin. The mode of caldesmon-induced profilactin dissociation was similar to that described for Mg2+, and 2 mM MgCl2 potentiated the caldesmon effect. Since both caldesmon and profilin have been found enriched in ruffling membranes of animal cells, our in vitro findings may be relevant to the regulation of actin filaments in living cells.     

The tail domain of myosin Va modulates actin binding to one head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.     

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

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

Analysis of tetramethylrhodamine-labeled actin polymerization and interaction with actin regulatory proteins

The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl beta,gamma-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.     

Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.     

Probing actin polymerization by intermolecular cross-linking

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.     

A model actin comet tail disassembling by severing

We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements.     

Connecting the dots: from actin polymerization to synapse formation

Protrusive behavior of dendritic spines on developing neurons has been previously suggested to mediate the formation of new axodendritic synaptic contacts. A study by Zito et al. in this issue of Neuron links actin polymerization in dendritic spines with the motility that the spines exhibit and the synapses that they form.     

Isolation and polymerization of brain actin.

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar to actin, is the major constituent. A method is described for isolating the "brain actin" to a purity of 90-95%. The isolation method begins with an extraction of bovine brain in low ionic strength buffer with ATP and sucrose. The extract is treated with NH4SO4, MgCl, and KCl and incubated at 37 degrees C. A precipitate is formed which contains primarily tubulin and brain actin. Resolubilization of the brain actin is achieved with a low ionic strength buffer solution with sucrose and ATP. Further purification is accomplished by a cycle of polymerization-depolymerization. This "brain actin" shares with muscle actin the following properties: (1) Similar molecular weight and molecular charge as determined by SDS polyacrylamide gel and ordinary disc electrophoresis; (2) Polymerization to a filamentous form under the same conditions; (3) Contains 3-methylhistidine; (4) Vinblastine sulfate will induce filament formation.