Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.     

Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium

The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin-actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1.     

ADP-ribosylated actin caps the barbed ends of actin filaments

The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.     

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Arabidopsis capping protein (AtCP) is a heterodimer that regulates assembly at the barbed ends of actin filaments

The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actin-binding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with Kd values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.     

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.     

Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4- mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on- rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.     

Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.     

Dynamic changes in the length distribution of actin filaments during polymerization can be modulated by barbed end capping proteins

During actin polymerization, it has been theorized that the actin filament length distribution initially grows in the form of a Gaussian before converting to produce that of an exponential. However, it has been difficult to demonstrate this experimentally. In this study, we use modern fluorescence microscopy techniques to observe the changing actin filament length distribution during and subsequent to the polymerization process. Nucleated actin filament growth using barbed end capping proteins (gelsolin and erythrocyte capping protein) leads to Gaussian length distributions that are relatively stable. As predicted, nucleated actin filament growth using actin/spectrin complexes follows a similar process until polymerization reaches equilibrium whereafter the Gaussian length distribution rapidly converts to that of an exponential. This study provides direct confirmation of the original theories for the mode of actin polymerization but raises doubts regarding the mechanism of the length distribution conversion from Gaussian to exponential.     

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.     

Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments

I measured the rate of elongation at the barbed and pointed ends of actin filaments by electron microscopy with Limulus sperm acrosomal processes as nuclei. With improvements in the mechanics of the assay, it was possible to measure growth rates from 0.05 to 280 s-1. At 22 degrees C in 1 mM MgCl2, 10 mM imidazole (pH 7), 0.2 mM ATP with 1 mM EGTA or 50 microM CaCl2 or with EGTA and 50 mM KCl, the elongation rates at both ends have a linear dependence on the ATP-actin concentration from the critical concentration to 20 microM. Consequently, over a wide range of subunit addition rates, the rate constants for association and dissociation of ATP-actin are constant. This shows that the nucleotide composition at or near the end of the growing filament is either the same over this range of growth rates or has no detectable effect on the rate constants. Under conditions where polymerization is fastest (MgCl2 + KCl + EGTA) the rate constants have these values: (table; see text) Compared with ATP-actin, ADP-actin associates slower at both ends, dissociates faster from the barbed end, but dissociates slower from the pointed end. Taking into account the events at both ends, these constants and a simple Oosawa-type model account for the complex three-phase dependence of the rate of polymerization in bulk samples on the concentration of ATP-actin monomers observed by Carlier, M.-F., D. Pantaloni, and E. D. Korn (1985, J. Biol. Chem., 260:6565-6571). These constants can also be used to predict the reactions at steady state in ATP. There will be slow subunit flux from the barbed end to the pointed end. There will also be minor fluctuations in length at the barbed end due to occasional rapid dissociation of strings of ADP subunits but the pointed end will be relatively stable.     

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.     

How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomer. Formed filaments keep a constant length of about 1 μm. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomers and can stabilize the pointed ends of actin filaments. We observed that tropornyosintroponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.     

Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

Ena/VASP proteins capture actin filament barbed ends

Ena/VASP (vasodialator-stimulated protein) proteins regulate many actin-dependent events, including formation of protrusive structures, fibroblast migration, neurite extension, cell-cell adhesion, and Listeria pathogenesis. In vitro, Ena/VASP activities on actin are complex and varied. They promote actin assembly, protect filaments from cappers, bundle filaments, and inhibit filament branching. To determine the mechanisms by which Ena/VASP proteins regulate actin dynamics at barbed ends, we monitored individual actin filaments growing in the presence of VASP and profilin using total internal reflection fluorescence microscopy. Filament growth was unchanged by VASP, but filaments grew faster in profilin-actin and VASP than with profilin-actin alone. Actin filaments were captured directly by VASP-coated surfaces via interactions with growing barbed ends. End-attached filaments transiently paused but resumed growth after becoming bound to the surface via a filament side attachment. Thus, Ena/VASP proteins promote actin assembly by interacting directly with actin filament barbed ends, recruiting profilin-actin, and blocking capping.     

Regulation of sodium channel activity by capping of actin filaments

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.     

Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin-actin complex.

The rate constant and equilibrium constant of association of an actin monomer with 1:1 gelsolin-actin complex isolated from chicken were measured by using fluorescently labeled actin. According to fluorescence stopped-flow experiments, the rate constant of formation of the 1:2 gelsolin-actin complex from 1:1 gelsolin-actin complex and actin was found to be about 2 x 10(7) M-1 s-1 under conditions where gelsolin binds Ca2+. The rate of dissociation of one actin molecule from the 1:2 gelsolin-actin complex was determined by exchange of actin for fluorescently labeled actin. The rate constant of dissociation was about 0.02 s-1. Thus, the equilibrium constant for association of actin with 1:1 gelsolin-actin complex can be calculated to be in the range of 10(9) M-1. The rate of dissociation of actin from 1:2 gelsolin-actin complex was independent of the Ca2+ concentration. Ca2+ affects only the rate of association of actin with 1:1 gelsolin-actin complex.     

The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.