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.     

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.     

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.     

The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1

The organization of actin filaments into large ordered structures is a tightly controlled feature of many cellular processes. However, the mechanisms by which actin filament polymerization is initiated from the available pool of profilin-bound actin monomers remain unknown in plants. Because the spontaneous polymerization of actin monomers bound to profilin is inhibited, the intervention of an actin promoting factor is required for efficient actin polymerization. Two such factors have been characterized from yeasts and metazoans: the Arp2/3 complex, a complex of seven highly conserved subunits including two actin-related proteins (ARP2 and ARP3), and the FORMIN family of proteins. The recent finding that Arabidopsis thaliana plants lacking a functional Arp2/3 complex exhibit rather modest morphological defects leads us to consider whether the large FORMIN family plays a central role in the regulation of actin polymerization. Here, we have characterized the mechanism of action of Arabidopsis FORMIN1 (AFH1). Overexpression of AFH1 in pollen tubes has been shown previously to induce abnormal actin cable formation. We demonstrate that AFH1 has a unique behavior when compared with nonplant formins. The activity of the formin homology domain 2 (FH2), containing the actin binding activity, is modulated by the formin homology domain 1 (FH1). Indeed, the presence of the FH1 domain switches the FH2 domain from a tight capper (Kd approximately 3.7 nM) able to nucleate actin filaments that grow only in the pointed-end direction to a leaky capper that allows barbed-end elongation and efficient nucleation of actin filaments from actin monomers bound to profilin. Another exciting feature of AFH1 is its ability to bind to the side and bundle actin filaments. We have identified an actin nucleator that is able to organize actin filaments directly into unbranched actin filament bundles. We suggest that AFH1 plays a central role in the initiation and organization of actin cables from the pool of actin monomers bound to profilin.     

The formin DAD domain plays dual roles in autoinhibition and actin nucleation

Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin beta(4). Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.     

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.     

Exogenous nucleation sites fail to induce detectable polymerization of actin in living cells

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Effect of capping protein on the kinetics of actin polymerization

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.     

Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets.

Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.     

Mechanism of actin filament nucleation

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.     

Actin dynamics at intracellular membranes: the Spir/formin nucleator complex

The assembly of actin monomers into filaments is a highly regulated basic cellular function. The structural organization of a cell, morphological changes or cell motility is dependent on actin filament dynamics. While within the last decade substantial knowledge has been acquired about actin dynamics at the cell membrane, today only little is known about the actin cytoskeleton and its functions at intracellular endosomal and organelle membranes. The Spir actin nucleators are specifically targeted towards endosomal membranes by a FYVE zinc finger membrane localization domain, and provide an important link to study the role of actin dynamics in the regulation of intracellular membrane transport. Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. Although Spir proteins can nucleate actin polymerization in vitro by themselves, they form a regulatory complex with the distinct actin nucleators of the formin subgroup (Fmn) of formins. A cooperative mechanism in actin nucleation has been proposed. Ongoing studies on the function and regulation of the Spir proteins in vesicle transport processes will reveal important insights into actin dynamics at intracellular membranes and how this regulates the highly directed and controlled routes of intracellular membrane trafficking.     

Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway

Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in aip1Δ mutant cells, lat A–insensitive actin assembly is significantly enhanced. We conclude that actin oligomer annealing is a physiologically relevant actin filament assembly pathway in vivo and identify Aip1 as a crucial factor for shifting the distribution of short actin oligomers toward monomers during disassembly.     

Emergence and maintenance of variable-length actin filaments in a limiting pool of building blocks

Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the length of single actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different lengths using the same set of molecular building blocks. Here, we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth-rate modulation by actin-binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneity. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning filamentous actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.     

Actin and myosin function in acanthamoeba

We have studied the functions of contractile proteins in Acanthamoeba by a combination of structural, biochemical and physiological approaches. We used electron microscopy and image processing to determine the three-dimensional structure of actin and the orientation of the molecule in the actin filament. We measured the rate constants for actin filament elongation and re-evaluated the effect of MgCl2 on the filament nucleation process. In Acanthamoeba actin polymerization is regulated, at least in part, by profilin, which binds to actin monomers, and by capping protein, which both nucleates polymerization and blocks monomer addition at the 'barbed' end of the filament. To test for physiological functions of myosin-II, we produced a monoclonal antibody that inhibits the actin-activated ATPase. When microinjected into living cells, this active-site-specific antibody inhibits amoeboid locomotion. We expect that similar experiments can be used to test for the physiological functions of the other components of the Acanthamoeba contractile system.     

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.     

Real-time observation of RecA filament dynamics with single monomer resolution

RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.