The effect of diffusion,depolymerization and nucleation promoting factors on actin gel growth

In eukaryotic cells, localized actin polymerization is able to deform the plasma membrane and push the cell forward. Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics. Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions. We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin-based movement of Listeria monocytogenes. We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control. Data analysis using an elastic model of actin gel growth gives an estimate of 2×10^–8 cm^–2 s^–1 for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cytoskeleton, providing corroboration of our model. The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated. Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex.     

Involvement of an arginine residue of actin in tropomyosin binding.

Using 1,2-cyclohexanedione, modification of three arginines per actin monomer in F-actin resulted in a loss of ability of the actin to interact with tropomyosin, although the F-actin polymer was not significantly depolymerized, the ability of the actin to activate the Mg²⁺-ATPase of myosin was not affected, and the secondary structure of the actin monomers was not appreciably altered. Isolation of peptides from a digest of modified F-actin indicated that the modified residues were Arg-28, Arg-95 and Arg-147. When actin was combined with tropomyosin prior to the modification treatment, Arg-95 was not modified, and the actin retained its ability to bind tropomyosin. These results therefore indicate a direct involvement of Arg-95 in the tropomyosin binding function of F-actin.     

Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate.     

Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments

We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a M_r value of 540 × 10³ as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 10³ dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm^?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a K_a value of 2 × 10⁶m^?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.     

The elasticity of spectrin-actin gels at high protein concentration

Human erythrocyte spectrin of high purity was studied alone and mixed with rabbit skeletal actin by dynamic rheometry as a function of protein concentration at pH 7.4 and 24 degrees C. Pure spectrin had a very low storage modulus, G', increasing slightly with increase in protein concentration (approximately 3 dynes/cm at 25 mg/ml). In contrast, unpurified cytoskeletal extracts containing spectrin, actin, and band 4.1 showed a marked concentration dependence for G', increasing to 150 dynes/cm at 20 mg/ml. Mixtures of purified spectrin and skeletal actin at a weight ratio of 4:1 also showed G' markedly dependent on concentration (approximately 150-200 dynes/cm at 20 mg/ml). Maximum elasticity of spectrin-actin gels occurred at a molar ratio of actin monomers to spectrin tetramers of 14:1. We conclude that the reconstituted in vitro spectrin-actin network consists of actin fibers cross-linked by spectrin tetramers at regular intervals. The gel is rapidly reformed after mechanical disruption or thermal collapse, indicating that the polymer fibers are in equilibrium with the constituent monomers.     

Brain spectrin fragments and crosslinks actin filaments

The effect of brain spectrin (fodrin) on actin has been studied using viscometry and fluorimetry. Brain spectrin resembles erythrocyte spectrin tetramer in its action on actin. Both proteins crosslink actin filaments giving rise to a large increase in the viscosity but fluorimetry shows that neither affects actin polymerization significantly. In addition, brain spectrin as well as erythrocyte spectrin fragments preformed actin filaments. Actin filaments incubated in the presence of either of the two proteins incorporate actin monomers at a much higher rate showing that more filament ends are generated.     

Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins

Actin and spectrin were isolated from washed red blood cell membranes. Spectrin bound and polymerized erythrocyte actin in the absence of potassium. Spectrin coated onto polystyrene latex particles bound 8–9 mol of erythrocyte actin per mol of spectrin when actin was in its depolymerized state. Spectrin enhanced the interaction of erythrocyte actin with muscle myosin as manifested by changes in Mg²⁺-ATPase activity. A similar enhancement also was observed with muscle α-actinin while muscle tropomyosin abolished these effects. The data suggest that spectrin may play the role of polymerizing factor as well as the anchoring site for erythrocyte actin just as α-actinin is the anchoring site for actin filaments in muscle and other non-muscle cells.     

Adsorption of actin at the air-water interface: a monolayer study

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging-plate method. Actin, a very soluble protein, moves from the subphase to the air-water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G-actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F-actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F-actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G-actin film at the beginning of the compression and of an F-actin film at the end of the compression process.     

Tropomyosin from human erythrocyte membrane polymerizes poorly but binds F-actin effectively in the presence and absence of spectrin

Actin in the human erythrocyte forms short protofilaments which are only long enough to accommodate tropomyosin monomers (Shen, B.W., Josephs, R. and Steck, T.L. (1986) J. Cell Biol. 102, 997-1006). This interaction between actin and tropomyosin monomers is predicted to be weak, since tropomyosin polymerization parallels its affinity for F-actin. We examine the binding of human erythrocyte tropomyosin to actin in the presence and absence of spectrin and its ability to polymerize. The binding of human erythrocyte tropomyosin to F-actin is not affected appreciably by the present of spectrin. Saturating F-actin with erythrocyte tropomyosin, however, weakens the binding of spectrin dimers to actin. Although tropomyosin from human erythrocyte and rabbit cardiac muscle have similar affinity for F-actin, the polymerizability of erythrocyte tropomyosin as determined by viscosity measurements is much reduced relative to muscle tropomyosin. This unusual property of erythrocyte tropomyosin is likely due to differences in its primary structure from other known tropomyosin at the amino and carboxyl terminal regions which are responsible for its head-to-tail polymerization and cooperative binding to F-actin. Analysis of the distribution of tyrosine by 2-dimensional tryptic mapping of 125I-labelled erythrocyte tropomyosin shows that tyrosine at positions 162, 214, 221, 261 and 267 in rabbit cardiac tropomyosin are conserved in human erythrocyte tropomyosin but Tyr-60 is absent. This observation suggests that erythrocyte tropomyosin has a carboxyl terminal region similar to its muscle counterparts but its amino terminal region resembles that of platelet tropomyosin which also lacks Tyr-60.     

High-affinity interaction of vinculin with actin filaments in vitro

Immunofluorescence and microinjection experiments have shown that vinculin (molecular weight 130,000) is localized at adhesion plaques of fibroblasts spread on a solid substrate. We found that this protein affects actin filament assembly and interactions in vitro at substoichiometric levels. Vinculin inhibits the rate of actin polymerization under conditions that limit nuclei formation, indicating an effect on the filament elongation step of the reaction. Vinculin also reduces actin filament-filament interaction measured with a low-shear viscometer. Scatchard plot analysis of the binding of ³H-labeled vinculin to actin filaments showed that there is one high-affinity binding site (dissociation constant = 20 nM) for every 1,500–2,000 actin monomers. These results suggest that vinculin interacts with a specific site located at the growing ends of actin filaments in a cytochalasin-like manner, a property consistent with its proposed function as a linkage protein between filaments and the plasma membrane.     

Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.     

Stabilization of actin by phalloidin: a differential scanning calorimetric study.

We have used differential scanning calorimetry to study the effects of phalloidin on F and G actin stability. For F actin, saturating concentrations of phalloidin induced an important shift on the transition temperature, Tm, from 69.5 degrees C to 83.5 degrees C. However, the calorimetric enthalpy remained unchanged. Using lower phalloidin concentrations, monomers linked to phalloidin, as well as neighboring unlinked monomers, were both stabilized. Contrary to previous reports, phalloidin was also shown to affect G actin, shifting its Tm from 59.5 degrees C to 75 degrees C. Two mechanisms are proposed to explain this finding: first, it could indicate a real interaction of phalloidin with G actin, and second, heating of the specimen during the scan could have induced polymerization of some G actin to the F form. The resulting F polymer would then interact with phalloidin, thus shifting the equilibrium between G and F actin towards the polymeric form.     

Defects in associating systems: example of actin.

A statistical mechanical model is given for linear associating systems that contain defects, using the double-stranded actin polymer as an example. We treat the system as a one-dimensional lattice that can desorb monomers (giving defects) using grand partition function techniques. The main difference from a standard adsorption problem is that the monomer units are also responsible for the structural integrity of the lattice (polymer) and if too many desorb the polymer will be broken. We use literature data to estimate the density of defects in the actin polymer.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.     

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca²⁺-dependent, but not completely reversible, as on removal of Ca²⁺ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca²⁺ in a micromolar range, with half maximum activation at about 3×10⁻⁶ M. The protein becomes inactivated when Ca²⁺ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.     

Comparison of filamin A-induced cross-linking and Arp2/3 complex-mediated branching on the mechanics of actin filaments.

We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 microm actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.     

Genetic studies of spectrin: new life for a ghost protein

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (β_H) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms.^(1,2) Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the β_H spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones. BioEssays 20:875–878, 1998. © 1998 John Wiley & Sons, Inc.     

Region of Longitudinal Growth in Striated Muscle Fibres

THE use of radioactively labelled amino-acids to locate the newly formed contractile proteins of muscle is not specific, for they are incorporated into all the proteins being synthesized by the muscle which contain that amino-acid in their sequence. We have attempted to label more specifically the newly formed actin subunits in growing muscle using radioactive adenosine. (We thank Dr M. R. Iyengar of the University of Pennsylvania for first making this suggestion.) The principle of the method is that the adenosine is incorporated into the structural ADP^1,2 of the actin monomers and the free adenosine and most other adenosine-containing compounds are removed from the muscle fibres by subsequent glycerol extraction³.     

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament "seeds" and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.     

Cooperativity in F-actin filaments on binding of myosin subfragments, demonstrated by fluorescence of 1, N6- ethenoadenosine diphosphate.

The fluorescence lifetime of 1,N⁶-ethenoadenosine diphosphate (?-ADP) is 33 ns when bound to F-actin at 4 °C. When heavy meromyosin or myosin subfragment-1 binds to the F-actin filament, the lifetime of ?-ADP drops, reaching 29 ns when every actin monomer is bound to a myosin head. The change in lifetime is a consequence of cooperative conformational changes among the actin monomers. The results of these experiments support the contention that there are differences in the ways in which the two heads of the myosin molecule interact with the actin filament.