The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition

Formin proteins are widely expressed in eukaryotes and play essential roles in assembling specific cellular actin-based structures. Formins are defined by a Formin Homology 2 (FH2) domain, as well as a proline-rich FH1 domain that binds the actin monomer binding protein, profilin, and other ligands. Constructs including FH2 of budding yeast Bni1 or fission yeast Cdc12 formins nucleate actin filaments in vitro. In this study, we demonstrate that FH2-containing constructs of murine mDia1 (also called p140 mDia or Drf1) are much more potent actin nucleators than the yeast formins. FH1 is necessary for nucleation when actin monomers are profilin bound. mDia1 is a member of the Diaphanous formin subfamily (Dia), whose members contain an N-terminal Rho GTPase binding domain (GBD) and a C-terminal Diaphanous autoinhibitory domain (DAD, ). Based on cellular and in vitro binding studies, an autoinhibitory model for Dia formin regulation proposes that GBD binding to DAD inhibits Dia-induced actin remodeling, whereas Rho binding activates by releasing GBD from DAD. Supporting this model, our results show that an N-terminal mDia1 construct strongly inhibits actin nucleation by the C terminus. RhoA partially relieves inhibition but does so when bound to either GDP or GTP analogs. Both N- and C-terminal mDia1 constructs appear to be multimeric.     

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.     

Crystal Structure of a Complex between Amino and Carboxy Terminal Fragments of mDia1: Insights into Autoinhibition of Diaphanous-Related Formins

Formin proteins direct the nucleation and assembly of linear actin filaments in a variety of cellular processes using their conserved formin homology 2 (FH2) domain. Diaphanous-related formins (DRFs) are effectors of Rho-family GTPases, and in the absence of Rho activation they are maintained in an inactive state by intramolecular interactions between their regulatory N-terminal region and a C-terminal segment referred to as the DAD domain. Although structures are available for the isolated DAD segment in complex with the interacting region in the N-terminus, it remains unclear how this leads to inhibition of actin assembly by the FH2 domain. Here we describe the crystal structure of the N-terminal regulatory region of formin mDia1 in complex with a C-terminal fragment containing both the FH2 and DAD domains. In the crystal structure and in solution, these fragments form a tetrameric complex composed of two interlocking N+C dimers. Formation of the tetramer is likely a consequence of the particular N-terminal construct employed, as we show that a nearly full-length mDia1 protein is dimeric, as are other autoinhibited N+C complexes containing longer N-terminal fragments. The structure provides the first view of the intact C-terminus of a DRF, revealing the relationship of the DAD to the FH2 domain. Delineation of alternative dimeric N+C interactions within the tetramer provides two general models for autoinhibition in intact formins. In both models, engagement of the DAD by the N-terminus is incompatible with actin filament formation on the FH2, and in one model the actin binding surfaces of the FH2 domain are directly blocked by the N-terminus.     

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.     

Formin proteins: a domain-based approach

Formin proteins are potent regulators of actin dynamics. Most eukaryotes have multiple formin isoforms, suggesting diverse cellular roles. Formins are modular proteins, containing a series of domains and functional motifs. The Formin homology 2 (FH2) domain binds actin filament barbed ends and moves processively as these barbed ends elongate or depolymerize. The FH1 domain influences FH2 domain function through binding to the actin monomer-binding protein, profilin. Outside of FH1 and FH2, amino acid similarity between formins decreases, suggesting diverse mechanisms for regulation and cellular localization. Some formins are regulated by auto-inhibition through interaction between the diaphanous inhibitory domain (DID) and diaphanous auto-regulatory domain (DAD), and activated by Rho GTPase binding to GTPase-binding domains (GBD). Other formins lack DAD, DID and GBD, and their regulatory mechanisms await elucidation.     

Crystal Structure of the Formin mDia1 in Autoinhibited Conformation

Background

Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).

Methodology/Principal Findings

Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia). The structure shows a tetrameric configuration (4 FH2 + 4 DID) in which the actin-binding sites on the FH2 domain are sterically occluded. However biochemical data suggest the full-length mDia1 is a dimer in solution (2 FH2 + 2 DID). Based on the crystal structure, we have generated possible dimer models and found that architectures of all of these models are incompatible with binding to actin filament but not to actin monomer. Furthermore, we show that the minimal functional monomeric unit in the FH2 domain, termed the bridge element, can be inhibited by isolated monomeric DID. NMR data on the bridge-DID system revealed that at least one of the two actin-binding sites on the bridge element is accessible to actin monomer in the inhibited state.

Conclusions/Significance

Our findings suggest that autoinhibition in the native DRF dimer involves steric hindrance with the actin filament. Although the structure of a full-length DRF would be required for clarification of the presented models, our work here provides the first structural insights into the mechanism of the DRF autoinhibition.     

Cloning and functional characterization of a formin-like protein (AtFH8) from Arabidopsis

The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.     

The lasso segment is required for functional dimerization of the Plasmodium formin 1 FH2 domain

Apicomplexan parasites, such as the malaria-causing Plasmodium species, utilize a unique way of locomotion and host cell invasion. This substrate-dependent gliding motility requires rapid cycling of actin between the monomeric state and very short, unbranched filaments. Despite the crucial role of actin polymerization for the survival of the malaria parasite, the majority of Plasmodium cellular actin is present in the monomeric form. Plasmodium lacks most of the canonical actin nucleators, and formins are essentially the only candidates for this function in all Apicomplexa. The malaria parasite has two formins, containing conserved formin homology (FH) 2 and rudimentary FH1 domains. Here, we show that Plasmodium falciparum formin 1 associates with and nucleates both mammalian and Plasmodium actin filaments. Although Plasmodium profilin alone sequesters actin monomers, thus inhibiting polymerization, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio. We have determined solution structures of P. falciparum formin 1 FH2 domain both in the presence and absence of the lasso segment and the FH1 domain, and show that the lasso is required for the assembly of functional dimers.     

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

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

New mechanisms and functions of actin nucleation

In cells the de novo nucleation of actin filaments from monomers requires actin-nucleating proteins. These fall into three main families--the Arp2/3 complex and its nucleation promoting factors (NPFs), formins, and tandem-monomer-binding nucleators. In this review, we highlight recent advances in understanding the molecular mechanism of actin nucleation by both well-characterized and newly identified nucleators, and explore current insights into their cellular functions in membrane trafficking, cell migration and division. The mechanisms and functions of actin nucleators are proving to be more complex than previously considered, with extensive cooperation and overlap in their cellular roles.     

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.     

Actin filament nucleation and elongation factors – structure–function relationships

The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tβ4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs–Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been “outsourced” to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.     

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

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

Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture

Formin proteins participate in a wide range of cytoskeletal processes in all eukaryotes. The defining feature of formins is a highly conserved approximately 400 residue region, the Formin Homology-2 (FH2) domain, which has recently been found to nucleate actin filaments. Here we report crystal structures of the S. cerevesiae Bni1p FH2 domain. The mostly alpha-helical FH2 domain forms a unique "tethered dimer" in which two elongated actin binding heads are tied together at either end by an unusual lasso and linker structure. Biochemical and crystallographic observations indicate that the dimer is stable but flexible, with flexibility between the two halves of the dimer conferred by the linker segments. Although each half of the dimer is competent to interact with filament ends, the intact dimer is required for actin nucleation and processive capping. The tethered dimer architecture may allow formins to stair-step on the barbed end of an elongating nascent filament.     

Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

The Rickettsia ～1800-amino-acid autotransporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments of Sca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.     

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.     

Regulation and targeting of the fission yeast formin cdc12p in cytokinesis

Formins are conserved actin nucleators which promote the assembly of actin filaments for the formation of diverse actin structures. In fission yeast Schizosaccharomyces pombe, the formin cdc12p is required specifically in assembly of the actin-based contractile ring during cytokinesis. Here, using a mutational analysis of cdc12p, we identify regions of cdc12p responsible for ring assembly and localization. Profilin-binding residues of the FH1 domain regulate actin assembly and processive barbed-end capping by the FH2 domain. Studies using photobleaching (FRAP) and sensitivity to latrunculin A treatment show that profilin binding modulates the rapid dynamics of actin and cdc12p within the ring in vivo. Visualized by functional GFP-fusion constructs expressed from the endogenous promoter, cdc12p appears in a small number of cytoplasmic motile spot structures that deliver the formin to the ring assembly site, without detectable formation of an intermediate band of "nodes." The FH3/DID region directs interphase spot localization, while an N-terminal region and the FH1-FH2 domains of cdc12p can target its localization to the ring. Mutations in putative DID and DAD regions do not alter regulation, suggesting that cdc12p is not regulated by a canonical autoinhibition mechanism. Our findings provide insights into the regulation of formin activity and the mechanisms of contractile ring dynamics and assembly.     

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.