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.     

Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.     

Identification of a Short Spir Interaction Sequence at the C-terminal End of Formin Subgroup Proteins

The actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2.Basic cell biological functions such as proliferation, migration, division, and vesicle transport rely on the organization of the actin cytoskeleton. The initiation of actin polymerization from free actin monomers is regulated by actin nucleation factors (NF),² which help to overcome the kinetic barrier of spontaneous G-actin nucleation and, thus, catalyze the formation of filamentous actin structures and networks (1). To date, three different classes of NFs are described, the ARP2/3 complex, FH2 domain containing NFs of the formin superfamily, and NFs containing one or multiple WH2 domains (Spire/Cordon-bleu/Leiomodin) (2). The formin superfamily is subdivided into seven subfamilies (Dia, FRL, DAAM, Delphilin, INF, FHOD, Fmn) (3). The mechanisms of actin nucleation as well as the regulation of the NFs vary significantly between the three classes (and also show variances in between the distinct superfamilies). Spire and Cappuccino are NFs that belong to the Spire subfamily of WH2 containing nucleators and to the Fmn subfamily of the FH2 domain containing formins, respectively. In contrast to the Arp2/3 complex that nucleates branched filaments, Spire and the formin Cappuccino nucleate unbranched actin filaments (4).Almost two decades ago it was found that mutants of the two Drosophila NFs (Spire/Cappuccino) have an identical phenotype in early Drosophila oogenesis, i.e. both induce premature ooplasmic streaming (5, 6). Later it was shown that both proteins cooperate in the generation of a dynamic actin mesh in the oocyte that prevents premature ooplasmic streaming (7). Spire and Cappuccino do not solely have the same mutant phenotype; the proteins also physically interact and cross-regulate each other. The Cappuccino C-terminal half, encoding the FH2 domain and flanking sequences, enhances the nucleation activity of Spire, whereas the nucleation activity of Cappuccino is decreased in the presence of the Spire-KIND domain (8).Cappuccino belongs to the Fmn subgroup of formins (3, 9). In mammals, two Fmn subgroup members (formin-1, formin-2) and two Spir proteins (Spir-1, Spir-2) exist (3, 10). The formin-2 and spir-1 genes are coexpressed in the developing and adult nervous system, and the proteins interact analogous to their Drosophila counterparts Spire and Cappuccino (8, 10). Several reports showed the importance of formin-2 in mouse oogenesis and here especially in the positioning of the meiotic spindle (11–14). Recently it was found that a dynamic actin mesh, as during Drosophila oogenesis, is also required for mouse oogenesis (11, 14). The correct localization of the meiotic spindle during mouse oogenesis and the resulting asymmetric division depends on an actin mesh that is built up by formin-2. Myosin-2 generates the pulling forces required for spindle movement (14). Beside the evolutionary conserved roles for the formins Cappuccino and formin-2, Spire family proteins also seem to be evolutionary conserved regulators of oocyte development. Spire genes of the African clawed frog Xenopus (pEg6) and the sea squirt Ciona savignyi (Pem-5) have been identified as maternal genes in the oocyte in analogy to its Drosophila homolog and are proposed to function in polarity during early embryogenesis (15, 16).In an initial characterization it was found that the KIND domains of Spir-1/dSpire interact with the C-terminal sequences of formin-2/Cappuccino, which encode the FH2 domains and flanking sequences (8). To gain a further understanding of the interaction and cross-regulation of the two proteins, we investigated this interaction in detail. The objective of the study was the dissection of the formin-2/Spir-1 interaction and the determination of the structural elements that are responsible for the binding. The dissection revealed a high affinity Spir-1 interaction site of formin-2, which could be mapped to the very C terminus of formin-2 adjacent to its core FH2 domain (formin Spir interaction (FSI) sequence). The FSI sequence is conserved among the members of the Fmn subgroup. Consistently we found that all mammalian members of the two distinct nucleator families, Spir-1/2 and Fmn-1/2, interact with each other.     

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.     

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.     

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.     

Analysis of the function of Spire in actin assembly and its synergy with formin and profilin

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.     

Regulatory interactions between two actin nucleators, Spire and Cappuccino

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.     

Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor

Formins are a conserved family of actin assembly-promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.     

Regulation of the Yeast Formin Bni1p by the Actin‐Regulating Kinase Prk1p

The formin family of proteins promotes the assembly of linear actin filaments in the cells of diverse eukaryotic organisms. The predominant formins in mammalian cells are self‐inhibited by an intramolecular interaction between two terminal domains and are activated by the binding of the Rho GTPases and other factors. In this study, we show that Bni1p, a formin required for the assembly of actin cables in budding yeast, is also regulated by an autoinhibitory mechanism and phosphorylation by the actin regulatory kinase Prk1p, and possibly Ark1p as well, plays a key role in unlocking the inhibition. Bni1p is phosphorylated by Prk1p at three [L/V/I]xxxxTG motifs in vitro, and the phosphorylation is sufficient to activate Bni1p by disrupting its intramolecular interaction. This finding extends the roles of Prk1p in the regulation of actin dynamics to be associated with both anterograde and retrograde transport pathways, i.e. exocytosis and endocytosis, in yeast.     

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.     

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.     

Molecular details of formin-mediated actin assembly

Formins are a large family of multi-domain polypeptides that form homodimers. The highly conserved formin homology 2 (FH2) domain and its neighboring formin homology 1 (FH1) domain, which are surrounded by regulatory domains, cooperate in rapidly assembling profilin-actin into long filaments while remaining continuously associated with the fast-growing barbed end. Recent biochemical, biophysical, theoretical and structural studies have concluded that diverse formins are mechanistically similar, but that the rates of various assembly states differ quantitatively, and have shed light on the mechanism of formin auto-regulation and activation by Rho GTPases.     

Phylogenetic analysis of the formin homology 2 domain

Formin proteins are key regulators of eukaryotic actin filament assembly and elongation, and many species possess multiple formin isoforms. A nomenclature system based on fundamental features would be desirable, to aid the rapid identification and characterization of novel formins. In this article, we attempt to systematize the formin family by performing phylogenetic analyses of the formin homology 2 (FH2) domain, an independently folding region common to all formins, which alone can influence actin dynamics. Through database searches, we identify 101 FH2 domains from 26 eukaryotic species, including 15 in mice. Sequence alignments reveal a highly conserved yeast-specific insert in the "knob loop" region of the FH2 domain, with unknown functional consequences. Phylogenetic analysis using minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML) algorithms strongly supports the existence of seven metazoan groups. Yeast FH2 domains segregate from all other eukaryotes, including metazoans, other fungi, plants, and protists. Sequence comparisons of non-FH2 regions support relationships between three metazoan groups (Dia, DAAM, and FRL) and examine previously identified coiled-coil and Diaphanous auto-regulatory domain sequences. This analysis allows for a formin nomenclature system based on sequence relationships, as well as suggesting strategies for the determination of biochemical and cellular activities of these proteins.     

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.     

The mouse formin, FRLalpha, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments

Formins are a conserved class of proteins expressed in all eukaryotes, with known roles in generating cellular actin-based structures. The mammalian formin, FRLalpha, is enriched in hematopoietic cells and tissues, but its biochemical properties have not been characterized. We show that a construct composed of the C-terminal half of FRLalpha (FRLalpha-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing. These multiple activities can be explained by a model in which FRLalpha-C binds filament sides but prefers the topology of sides at the barbed end (end-sides) to those within the filament. This preference allows FRLalpha-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend. Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding model for formins. Profilin partially relieves barbed end elongation inhibition by FRLalpha-C. When non-muscle actin is used, FRLalpha-C's effects are largely similar. FRLalpha-C's ability to sever filaments is the first such activity reported for any formin. Because we find that mDia1-C does not sever efficiently, severing may not be a property of all formins.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

Expression of multiple formins in adult tissues and during developmental stages of mouse brain

Formins are highly conserved heterogeneous family of proteins with several isoforms having significant contribution in multiple cellular functions. Formins play crucial role in remodelling of actin cytoskeleton and thus play important role in cell motility. Formins are also involved in many cellular activities like determining cell polarity, cytokinesis and morphogenesis. Formins are multi domain protein with characteristic homodimeric formin homology 2 (FH2) domain. It nucleates the actin filaments and its activity is regulated by the presence of characteristic formin homology 1 (FH1) domain. In higher mammals like human and mouse fifteen different formin isoforms are present. However the function and expression pattern of each and every formin in different adult tissues are not well characterized. Here we have found that multiple formins are expressing in each adult tissue of mouse, irrespective of their origin from the germ layer. Formins are also expressing from early stage of development to the adulthood in brain. The expression of many formins in a single tissue of adult mouse indicates that regulation of actin cytoskeleton dynamics by formins may be crucial for physiological processes like wound healing, tissue repairing, exocytosis, endocytosis, synapse formation and maintenance. Expression of FMNL2 and Fhdc1 are high in adult mouse brain as compare to embryonic stages. Higher expression of FMNL2 and Fhdc1 indicates that FMNL2 and Fhdc1 might be very important for the adult brain functions.     

Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

Background  

Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures.