Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament

The balance between dynamic and stable actin filaments is essential for the regulation of cellular functions including the determination of cell shape and polarity, cell migration, and cytokinesis. Proteins that regulate polymerization at the filament ends and filament stability confer specificity to actin filament structure and cellular function. The dynamics of the barbed, fast-growing end of the filament are controlled in space and time by both positive and negative regulators of actin polymerization. Capping proteins inhibit the addition and loss of subunits, whereas other proteins, including formins, bind at the barbed end and allow filament growth. In this work, we show that tropomyosin regulates dynamics at the barbed end. Tropomyosin binds to constructs of FRL1 and mDia2 that contain the FH2 domain and modulates formin-dependent capping of the barbed end by relieving inhibition of elongation by FRL1-FH1FH2, mDia1-FH2, and mDia2-FH2 in an isoform-dependent fashion. In this role, tropomyosin functions as an activator of formin. Tropomyosin also inhibits the binding of FRL1-FH1FH2 to the sides of actin filaments independent of the isoform. In contrast, tropomyosin does not affect the ability of capping protein to block the barbed end. We suggest that tropomyosin and formin act together to ensure the formation of unbranched actin filaments, protected from severing, that could be capped in stable cellular structures. This role, in addition to its cooperative control of myosin function, establishes tropomyosin as a universal regulator of the multifaceted actin cytoskeleton.     

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing

INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

An Arabidopsis Class II Formin,AtFH19, Nucleates Actin Assembly,Binds to the Barbed End of Actin Filaments and Antagonizes the Effect of AtFH1 on Actin Dynamics

Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes: there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFH1. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FH1FH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FH1FH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.     

The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin.     

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.     

INF2 Is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization

Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed "diaphanous formins" are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory domain (DID) and a C-terminal diaphanous autoregulatory domain (DAD). Here, we found several striking features for the mouse formin, INF2. First, INF2 interacted directly with actin through a region C-terminal to the FH2. This second interacting region sequesters actin monomers, an activity that is dependent on a WASP homology 2 (WH2) motif. Second, the combination of the FH2 and C-terminal regions of INF2 resulted in its curious ability to accelerate both polymerization and depolymerization of actin filaments. The mechanism of the depolymerization activity, which is novel for formin proteins, involves both the monomer binding ability of the WH2 and a potent severing activity that is dependent on covalent attachment of the FH2 to the C terminus. Phosphate inhibits both the depolymerization and severing activities of INF2, suggesting that phosphate release from actin subunits in the filament is a trigger for depolymerization. Third, INF2 contains an N-terminal DID, and the WH2 motif likely doubles as a DAD in an autoinhibitory interaction.     

Formins regulate actin filament flexibility through long range allosteric interactions

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.     

Conformational changes in actin filaments induced by formin binding to the barbed end

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.     

Interaction of the N- and C-terminal autoregulatory domains of FRL2 does not inhibit FRL2 activity

Formin homology proteins are a highly conserved family of cytoskeletal remodeling proteins best known for their ability to induce the formation of long unbranched actin filaments. They accomplish this by nucleating the de novo polymerization of F-actin and also by acting as F-actin barbed end "leaky cappers" that allow filament elongation while antagonizing the function of capping proteins. More recently, it has been reported that the FH2 domains of FRL1 and mDia2 and the plant formin AFH1 are able to bind and bundle actin filaments via distinct mechanisms. We find that like FRL1, FRL2 and FRL3 are also able to bind and bundle actin filaments. In the case of FRL3, this activity is dependent upon a proximal DAD/WH2-like domain that is found C-terminal to the FH2 domain. In addition, we show that, like other Diaphanous-related formins, FRL3 activity is subject to autoregulation mediated by the interaction between its N-terminal DID and C-terminal DAD. In contrast, the DID and DAD of FRL2 also interact in vivo and in vitro but without inhibiting FRL2 activity. These data suggest that current models describing DID/DAD autoregulation via steric hindrance of FH2 activity must be revised. Finally, unlike other formins, we find that the FH2 and N-terminal dimerization domains of FRL2 and FRL3 are able to form hetero-oligomers.     

The Uncoupling of the Effects of Formins on the Local and Global Dynamics of Actin Filaments

In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.     

Mechanisms of formin-mediated actin assembly and dynamics

Cellular viability requires tight regulation of actin cytoskeletal dynamics. Distinct families of nucleation-promoting factors enable the rapid assembly of filament nuclei that elongate and are incorporated into diverse and specialized actin-based structures. In addition to promoting filament nucleation, the formin family of proteins directs the elongation of unbranched actin filaments. Processive association of formins with growing filament ends is achieved through continuous barbed end binding of the highly conserved, dimeric formin homology (FH) 2 domain. In cooperation with the FH1 domain and C-terminal tail region, FH2 dimers mediate actin subunit addition at speeds that can dramatically exceed the rate of spontaneous assembly. Here, I review recent biophysical, structural, and computational studies that have provided insight into the mechanisms of formin-mediated actin assembly and dynamics.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

The filamentous actin cross-linking/bundling activity of mammalian formins

Formins are multidomain proteins that regulate actin filament dynamics and are defined by the formin homology 2 domain. Biochemical assays suggest that mammalian formins display actin-filament nucleation, severing, and bundling activities. Whether formins can cross-link actin filaments into viscoelastic arrays and the effectiveness of formins' bundling activity compared with that of important filamentous actin (F-actin) cross-linking/bundling proteins are unknown. Here, we used rigorous in vitro rheologic assays to deconvolve the dynamic cross-linking activity from the bundling activity of formin FRL1 and the closely related mDia1 and mDia2. In addition, we compared these formins with the canonical F-actin bundling protein fascin and cross-linking/bundling proteins alpha-actinin and filamin. We found that FRL1 and mDia2, but not mDia1, can help F-actin form highly elastic networks. FRL1 and mDia2 mediate the formation of highly elastic F-actin networks as effectively and rapidly as alpha-actinin and filamin but only past a relatively high actin-to-formin molar ratio of 50:1. Past that threshold molar ratio, the mechanical properties of F-actin/formin networks are independent of formin concentration, similar to fascin. Moreover, unlike those for alpha-actinin and filamin but similar to those for fascin, F-actin/formin networks show no strain-induced hardening. mDia1 cannot bundle F-actin but can weakly cross-link filaments at high concentrations. Point mutagenesis reveals that reducing the barbed-end binding activity of FRL1 and mDia2 greatly enhances the rate of formation of F-actin gels but does not significantly affect the mechanical properties of the resulting networks at steady state. Together, these results suggest that the mechanical behaviors of FRL1 and mDia2 are fundamentally different from those of cross-linking/bundling proteins alpha-actinin and filamin but qualitatively similar to the mechanical behavior of the bundling protein fascin, albeit with a dramatically increased (>10-fold) threshold concentration for transition to bundling, which nevertheless leads to much stiffer F-actin networks than fascin.     

Mechanistic differences in actin bundling activity of two mammalian formins, FRL1 and mDia2

Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.     

Assembly and Turnover of Short Actin Filaments by the Formin INF2 and Profilin

INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.     

Control of the assembly of ATP- and ADP-actin by formins and profilin

Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.     

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.     

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.