Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1

BACKGROUND: Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo. RESULTS: Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo. CONCLUSIONS: We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.     

Identification of Arabidopsis cyclase-associated protein 1 as the first nucleotide exchange factor for plant actin

The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP-actin.     

Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks

BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.     

Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of profilin on actin-bound nucleotide exchange and actin polymerization dynamics

We have investigated the effects of profilin on nucleotide binding to actin and on steady state actin polymerization. The rate constants for the dissociation of ATP and ADP from monomeric Mg-actin at physiological conditions are 0.003 and 0.009 s-1, respectively. Profilin increases these dissociation rate constants to 0.08 s-1 for MgATP-actin and 1.4 s-1 for MgADP-actin. Thus, profilin can increase the rate of exchange of actin-bound ADP for ATP by 140-fold. The affinity of profilin for monomeric actin is found to be similar for MgATP-actin and MgADP-actin. Continuous sonication was used to allow study of solutions having sustained high filament end concentrations. During sonication at steady state, F-actin depolymerizes toward the critical concentration of ADP-actin [Pantaloni, D., et al. (1984)J. Biol. Chem. 259, 6274-6283], our analysis indicates that under these conditions a significant number of filaments contain terminal ADP-actin subunits. Addition of profilin to this system increases the polymer concentration and increases the steady state ATPase activity during sonication. These data are explained by the fast exchange of ATP for ADP on the profilin-ADP-actin complex, resulting in rapid ATP-actin regeneration. An important function of profilin may be to provide the growing ends of filaments with ATP-actin during periods when the monomer cycling rate exceeds the intrinsic nucleotide exchange rate of monomeric actin.     

Random copolymerization of ATP-actin and ADP-actin

The equilibrium of the copolymerization of ATP-actin and ADP-actin was investigated by an analysis of the critical concentrations of mixtures of ATP-actin and ADP-actin. The molar ratio of bound ATP to bound ADP was controlled by the ratio of free ATP and ADP. The experiments were performed under conditions (100 mM KCl, l mM MgCl2, pH 7.5, 25 degrees C) where the ATP hydrolysis following binding of actin monomers to barbed filament ends was so slow that the distribution of ATP or ADP bound to the subunits near the ends of filaments was not affected by ATP hydrolysis. According to the analysis of the critical concentrations, the equilibrium constants for incorporation of ATP-actin or ADP-actin into filaments were independent of the type of nucleotide bound to contiguous subunits.     

Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.     

Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility

Twinfilins are conserved actin-binding proteins composed of two actin depolymerizing factor homology (ADF-H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP-G-actin and caps filament barbed ends with preferential affinity for ADP-bound ends. Twinfilin replaces capping protein and promotes motility of N-WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilin's function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP-actin-sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin-functionalized beads. The isolated ADF-H domains do not cap barbed ends nor promote motility, but sequester ADP-actin, the C-terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF-H domains and gelsolin segments.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.     

The dynamic instability of actin filament barbed ends

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

IntroductionIn cells, actin assembles into filament networks with diverse architectures and lifetimes, playing key roles in functions such as endocytosis, cell motility, and cell division. These filament networks are maintained and renewed by actin turnover, which implies that assembly and disassembly must take place simultaneously and in a controlled manner within the networks. Each actin filament end has the ability to either grow or shrink, depending on the concentration of actin and regulatory proteins, but pure actin treadmills at steady state: ATP-actin is added at the barbed end at a rate matching the departure of ADP-actin from the pointed end, and ATP hydrolysis takes place within the filament. This hallmark feature of actin dynamics has been known for decades (Wegner, 1976) and has been generalized to the cell context, in which it is commonly assumed that actin polymerization takes place at the barbed end, while depolymerization takes place only at the pointed end (whether it be the ends of filaments within the network or the ends of fragments that have detached from it). This notion is reinforced by the fact that the cytoplasm contains high concentrations of monomeric actin (G-actin) in complex with profilin (Funk et al., 2019), which is unable to bind to pointed ends and should drive the elongation of all noncapped barbed ends.Recently, however, in vitro studies have identified two seemingly independent mechanisms in which, in the presence of profilin-actin, filament barbed ends alternate between phases of growth and depolymerization. This behavior, referred to as “dynamic instability,” is widely observed for microtubules but was unexpected for actin filaments. It suggests that cells could use barbed ends for both elongation and disassembly.Driving the depolymerization of barbed ends with cofilin side-decorationProteins of the actin depolymerizing factor (ADF)/cofilin family (henceforth cofilin) are composed of a single ADF-homology (ADF-H) domain and are mostly known for their actin filament–severing activity (De La Cruz, 2009). Cofilin binds cooperatively to the sides of actin filaments, forming clusters where the conformation of the filament is locally altered, leading to its severing at cofilin cluster boundaries. In addition, the barbed ends of cofilin-decorated filaments steadily depolymerize, despite the presence of G-actin and profilin-actin (Fig. 1 A) and even capping protein (CP) in solution (Wioland et al., 2017, 2019). This unexpected result likely originates from the conformational change of actin subunits at the barbed end, induced by cofilin side-binding. As a consequence, filaments exposed to G-actin (with or without profilin), CP, and cofilin alternate between phases of barbed-end elongation and barbed-end depolymerization. In these conditions, actin filament barbed ends thus exhibit a form of dynamic instability.Open in a separate windowFigure 1.Two mechanisms that give rise to barbed-end depolymerization in elongation-promoting conditions. (A) When a cofilin side-decorated region reaches the barbed end, adding a new actin or profilin-actin becomes very difficult, and the barbed end depolymerizes. Not represented: Capping by CP can lead to depolymerization, as it allows the cofilin cluster to reach the barbed end, which then has a much weaker affinity for CP and steadily depolymerizes. Also, severing events occur at cofilin cluster boundaries, creating new barbed ends, either bare or cofilin-decorated. (B) Twinfilin binds to the barbed end, preventing its elongation and causing its depolymerization. Whether twinfilin remains processively attached to the depolymerizing barbed end or departs with the actin subunits is still unknown. Twinfilin has no impact on the elongation of mDia1-bearing barbed ends.Driving the depolymerization of barbed ends with twinfilin end-targetingTwinfilin has two ADF-H domains, but unlike cofilin, it binds poorly to the sides of actin filaments. Rather, twinfilin appears to mainly sequester ADP-actin monomers and target the barbed end to modulate its elongation and capping. Recent in vitro studies have shown that the interaction of twinfilin with actin filament barbed ends could drive their depolymerization, even in the presence of G-actin and profilin-actin (Johnston et al., 2015; Hakala et al., 2021; Shekhar et al., 2021). Very interestingly, the processive barbed-end elongator formin mDia1 is able to protect barbed ends from twinfilin, allowing them to sustain elongation (Shekhar et al., 2021). This leads to a situation in which, as filaments are exposed to profilin-actin and twinfilin, mDia1-bearing barbed ends elongate while bare barbed ends depolymerize (Fig. 1 B). It is safe to assume that, if filaments were continuously exposed to this protein mix including formin in solution, they would alternate between phases of growth and shrinkage over time, as formins come on and fall off the barbed end. This mix of proteins would therefore constitute another situation causing actin filament dynamic instability.From actin treadmilling to dynamic instability, in cells?This newly identified versatile behavior of actin filaments is reminiscent of microtubules. While dynamic instability is the hallmark behavior of microtubules, they can also be made to treadmill steadily by adding 4 microtubule-associated proteins (Arpağ et al., 2020). In cells, both microtubule dynamic instability and treadmilling have been clearly observed (Wittmann et al., 2003). In contrast, the disassembly of single actin filaments, either embedded in a network or severed from it, has not yet been directly observed in cells. Despite insights from techniques such as single-molecule speckle microscopy, it is still unclear from which end actin filaments depolymerize, even in networks that appear to globally treadmill, such as the lamellipodium. Pointed end depolymerization alone cannot account for what is observed in cells (Miyoshi et al., 2006) and alternative mechanisms have been proposed, including brutal filament-to-monomer transitions occurring in bursts, driven by cofilin, coronin, and Aip1 (Brieher, 2013; Tang et al., 2020).In cells, the high amounts of available G-actin (tens of micromolars; Funk et al., 2019) should limit barbed-end depolymerization. Based on the reported on-rate for ATP–G-actin at the barbed ends of cofilin-decorated filaments (Wioland et al., 2017, 2019), we can estimate that these barbed ends, under such conditions, would depolymerize for tens of seconds before being “rescued,” which is enough to remove tens of subunits from each filament. In contrast, twinfilin concentrations similar to those of G-actin appear necessary to drive barbed-end depolymerization (Hakala et al., 2021; Shekhar et al., 2021). As proteomics studies in HeLa cells report that twinfilin is 50-fold less abundant than actin, this may be difficult to achieve in cells (Bekker-Jensen et al., 2017). However, future studies may uncover proteins, or posttranslational modifications of actin, that enhance the ability of twinfilin to drive barbed-end depolymerization in the presence of high concentrations of profilin-actin.Molecular insights and possible synergiesWhile cofilin and twinfilin both interact with actin via ADF-H domains, they appear to drive barbed-end depolymerization through different mechanisms: twinfilin by directly targeting the barbed end, and cofilin by decorating the filament sides, thereby changing the conformation of the filament and putting its barbed end in a depolymerization-prone state.The two mechanisms, nonetheless, share clear similarities. For instance, cofilin side-binding and twinfilin end-targeting both slow down ADP-actin barbed-end depolymerization, compared with bare ADP-actin filaments (Wioland et al., 2017; Hakala et al., 2021; Shekhar et al., 2021). Strikingly, a crystal structure of the actin/twinfilin/CP complex indicates that the actin conformational change induced by twinfilin binding at the barbed end is similar to that induced by cofilin decorating the sides (Mwangangi et al., 2021). It is thus possible that the dynamic instability of actin filament barbed ends reflects the same conformation changes, triggered either by cofilin side-decoration or twinfilin end-targeting.In addition to decorating the filament sides, cofilin targets ADP-actin barbed ends. Unlike twinfilin, the direct interaction of cofilin with the barbed end cannot cause its depolymerization in the presence of ATP-actin monomers. Indeed, cofilin end-targeting accelerates the depolymerization of ADP-actin barbed ends in the absence of G-actin, but cofilin does not appear to interact with growing ATP-actin barbed ends (Wioland et al., 2017). This is in stark contrast with twinfilin end-targeting, which slows down ADP-actin depolymerization and accelerates ADP–Pi-actin depolymerization (Shekhar et al., 2021). These different behaviors regarding the nucleotide state of actin are intriguing and should be investigated further.Cofilin thus needs to decorate the filament sides in order to have an impact on barbed-end dynamics in elongation-promoting conditions. However, it is unknown whether cofilin side-decoration extends all the way to the terminal subunits and occupies sites that twinfilin would target. Thus, it is unclear whether cofilin and twinfilin would compete or synergize to drive barbed-end depolymerization.Synergies with other proteins are also worth further investigation, CP being an interesting candidate. Cofilin side-decoration drastically decreases the barbed-end affinity for CP, and capped filaments are thereby an efficient intermediate to turn growing barbed ends into depolymerizing barbed ends (Wioland et al., 2017). Twinfilin interacts with CP and the barbed end to enhance uncapping (Hakala et al., 2021; Mwangangi et al., 2021). Since CP can bind mDia1-bearing barbed ends and displace mDia1 (Bombardier et al., 2015; Shekhar et al., 2015), perhaps CP can also contribute to turn growing, mDia1-bearing barbed ends into depolymerizing barbed ends, by removing mDia1 from barbed ends and subsequently getting displaced from the barbed end by twinfilin.Finally, it is worth noting that profilin, which does not contain an ADF-H domain, also interacts with the barbed face of G-actin and with the barbed end of the filament. When profilin is in sufficient excess, it is able to promote barbed-end depolymerization in the presence of ATP–G-actin (Pernier et al., 2016). Unlike twinfilin, its depolymerization-promoting activity is not prevented by formin mDia1, and it thus does not lead to dynamic instability (bare and mDia1-bearing barbed ends all either grow or depolymerize). The coexistence of growing, mDia1-bearing barbed ends and depolymerizing, twinfilin-targeted barbed ends (Fig. 1 B) was observed in the presence of profilin (Shekhar et al., 2021), but profilin actually may not be required. Future studies should determine the exact role of profilin in this mechanism.ConclusionThe extent to which barbed-end dynamic instability contributes to actin turnover in cells is not known, but possible molecular mechanisms have now been identified. They should change the way we envision actin network dynamics, as we must now consider the possibility that cells also exploit the barbed end for disassembly. More work is needed to further document these mechanisms, but the idea of a “generalized treadmilling” has now been contradicted at its source: in vitro experiments.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.     

Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation

The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize [Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166-4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213-218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806-8812]. This simple model fails to predict two new experimental observations made with Acanthamoeba actin in 50 mM KC1, 1 mM MgCl2, and 1 mM EGTA. First, Acanthamoeba profilin inhibits elongation of actin filaments far more at the pointed end than at the barbed end. According, to the simple model, the Kd for the profilin-actin complex is less than 5 microM on the basis of observations at the pointed end and greater than 50 microM for the barbed end. Second, profilin inhibits nucleation more strongly than elongation. According to the simple model, the Kd for the profilin-actin complex is 60-140 microM on the basis of two assays of elongation but 2-10 microM on the basis of polymerization kinetics that reflect nucleation. These new findings can be explained by a new and more complex model for the mechanism of action that is related to a proposal of Tilney and co-workers [Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113-124]. In this model, profilin can bind both to actin monomers with a Kd of about 5 microM and to the barbed end of actin filaments with a Kd of about 50-100 microM. An actin monomer bound to profilin cannot participate in nucleation or add to the pointed end of an actin filament. It can add to the barbed end of a filament. When profilin is bound to the barbed end of a filament, actin monomers cannot bind to that end, but the terminal actin protomer can dissociate at the usual rate. This model includes two different Kd's--one for profilin bound to actin monomers and one for profilin bound to an actin molecule at the barbed end of a filament. The affinity for the end of the filament is lower by a factor of 10 than the affinity for the monomer, presumably due to the difference in the conformation of the two forms of actin or to steric constraints at the end of the filament.     

Polymerization of ADP-actin

Using hexokinase, glucose, and ATP to vary reversibly the concentrations of ADP and ATP in solution and bound to Acanthamoeba actin, I measured the relative critical concentrations and elongation rate constants for ATP-actin and ADP-actin in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM nucleotide, 0.1 mM CaCl2, 10 mM imidazole, pH 7. By both steady-state and elongation rate methods, the critical concentrations are 0.1 microM for ATP-actin and 5 microM for ADP-actin. Consequently, a 5 microM solution of actin can be polymerized, depolymerized, and repolymerized by simply cycling from ATP to ADP and back to ATP. The critical concentrations differ, because the association rate constant is 10 times higher and the dissociation rate constant is five times lower for ATP-actin than ADP-actin. These results show that ATP-actin occupies both ends of actin filaments growing in ATP. The bound ATP must be split on internal subunits and the number of terminal subunits with bound ATP probably depends on the rate of growth.     

Effects of macrophage profilin on actin in the presence and absence of acumentin and gelsolin

We purified profilin from rabbit alveolar macrophages and documented its structural and functional similarity to profilins isolated from other cells. The KD for formation of the macrophage profilin-actin complex was 3.0 +/- 0.8 microM (mean +/- S.D.). The affinity of this protein for actin did not change significantly in the presence of various concentrations of KCl and MgCl2, profilin-actin complex concentration being strictly dependent on the critical actin monomer concentration and free profilin concentration. We also examined profilin's interactions with actin in the presence of acumentin, a macrophage protein which inhibits actin monomer exchange at the "pointed" ends of actin filaments. Low concentrations of this protein caused substantial decreases in estimated profilin-actin complex concentration. The macrophage gelsolincalcium ion complex which blocks exchange at the "barbed" end of actin filaments, when added to profilin and actin solutions in substoichiometric concentrations, caused large increases in estimated profilin-actin complex concentration. The changes in calculated profilin-actin complex concentration induced by these two actin-modulating proteins were too large to be explained solely by their effects on critical actin monomer concentration.     

Structure and Mechanism of Mouse Cyclase-associated Protein (CAP1) in Regulating Actin Dynamics

Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding β-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.     

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.     

A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin

Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.     

Mammalian and Malaria Parasite Cyclase-associated Proteins Catalyze Nucleotide Exchange on G-actin through a Conserved Mechanism

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.     

ATP hydrolysis by the gelsolin-actin complex and at the pointed ends of gelsolin-capped filaments

To obtain kinetic information about the pointed ends of actin filaments, experiments were carried out in the presence of gelsolin which blocks all events at the kinetically dominant barbed ends. The 1:2 gelsolin-actin complex retains 1 mol/mol of actin-bound ATP, but it neither hydrolyzes the ATP nor exchanges it with ATP free in solution at a significant rate. On the other hand, the actin filaments with their barbed ends capped with gelsolin hydrolyze ATP relatively rapidly at steady state, apparently as a result of the continued interaction of ATP-G-actin with the pointed ends of the filaments. ATP hydrolysis during spontaneous polymerization of actin in the presence of relatively high concentrations of gelsolin lags behind filament elongation so that filaments consisting of as much as 50% ATP-actin subunits are transiently formed. Probably for this reason, during polymerization the actin monomer concentration transiently reaches a concentration lower than the final steady-state critical concentration of the pointed end. At steady state, however, there is no evidence for an ATP cap at the pointed ends of gelsolin-capped filaments, which differs from the barbed ends which do have an ATP cap in the absence of gelsolin. As there is no reason presently to think that gelsolin has any effect on events at the pointed ends of filaments, the properties of the pointed ends deduced from these experiments with gelsolin-capped filaments are presumably equally applicable to the pointed ends of filaments in which the barbed ends are free.