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PubMed2003

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.

Actins↗
PubMed2006

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.

Actin Cytoskeleton↗
PubMed2002

Yeast formins regulate cell polarity by controlling the assembly of actin cables.

Formins are conserved Rho-GTPase effectors that communicate Rho-GTPase signals to the cytoskeleton. We found that formins were required for the assembly of one of the three budding yeast actin structures: polarized arrays of actin cables. A dominant-active formin induced the assembly of actin cables. The activation and localization of the formin Bni1p required components of the polarisome complex. These findings potentially define the cellular function of formins in budding yeast and explain their involvement in the generation of cell polarity. A requirement for formins in constructing specific actin structures might be the basis for the diverse activities of formins in development.

Actins↗
PubMed2004

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.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Actin polymerization upon processive capping by formin: a model for slowing and acceleration.

Formin family proteins act as processive cappers of actin filaments, and determine the dynamics of a number of intracellular processes that are based on actin polymerization. The rate of filament growth upon processive capping varies within a broad range depending on the formin type and presence of profilin. While FH2 domains of various formins slow down polymerization by different extents, the FH1-FH2 domains in conjunction with profilin accelerate the reaction. Study of the physical mechanism of processive capping is vital for understanding the intracellular actin dynamics. We propose a model predicting that variation of a single physical parameter-the effective elastic energy of the formin-capped barbed end-results in the observed diversity of the polymerization rates. The model accounts for the whole range of the experimental results including the drastic slowing down of polymerization by FH2 of Cdc12 formin and the 4.5-fold acceleration of the reaction by FH1-FH2 of mDai1 formin in the presence of profilin. Fitting the theoretical predictions to the experimental curves provides the values of the effective elastic energies of different formin-barbed end complexes.

Actin Cytoskeleton↗
PubMed2006

Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2.

Formin family proteins coordinate actin filaments and microtubules. The mechanisms by which formins bind and regulate the actin cytoskeleton have recently been well defined. However, the molecular mechanism by which formins coordinate actin filaments and microtubules remains poorly understood. We demonstrate here that Isoform-Ib of the Formin-1 protein (Fmn1-Ib) binds to microtubules via a protein domain that is physically separated from the known actin-binding domains. When expressed at low levels in NIH3T3 fibroblasts, Fmn1-Ib protein localizes to cytoplasmic filaments that nocodazole disruption confirmed as interphase microtubules. A series of progressive mutants of Fmn1-Ib demonstrated that deletion of exon-2 caused dissociation from microtubules and a stronger association with actin membrane ruffles. The exon-2-encoded peptide binds purified tubulin in vitro and is also sufficient to localize GFP to microtubules. Exon-2 does not contain any known formin homology domains. Deletion of exon 5, 7, 8, the FH1 domain or FH2 domain did not affect microtubule binding. Thus, our results indicate that exon-2 of Fmn1-Ib encodes a novel microtubule-binding peptide. Since formin proteins associate with actin filaments through the FH1 and FH2 domains, binding to interphase microtubules through this exon-2-encoded domain provides a novel mechanism by which Fmn1-Ib could coordinate actin filaments and microtubules.

Actins↗
PubMed2004

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.

Actins↗
PubMed1999

Gli3 (Xt) and formin (ld) participate in the positioning of the polarising region and control of posterior limb-bud identity.

During initiation of limb-bud outgrowth in vertebrate embryos, the polarising region (limb-bud organizer) is established upon activation of the Sonic Hedgehog (SHH) signaling molecule at the posterior limb-bud margin. Another hallmark of establishing anteroposterior limb-bud identities is the colinear activation of HoxD genes located at the 5' end of the cluster (5'HoxD genes). The unique and shared functions of Gli3 and formin in these determinative events were genetically analyzed using single and double homozygous Extra-toes (Xt; disrupting Gli3) and limb deformity (ld; disrupting formin) mouse embryos. Analysis of the limb skeletal phenotypes reveals genetic interaction of the two genes. In addition to loss of digit identity and varying degrees of polydactyly, proximal skeletal elements are severely shortened in Xt;ld double homozygous limbs. The underlying molecular defects affect both establishment of the polarising region and posterior limb-bud identity. In particular, the synergism between Gli3- and formin-mediated mesenchyme-AER interactions positions the SHH signaling center at the posterior limb-bud margin. The present study shows that establishment and positioning of the polarising region is regulated both by restriction of Shh through Gli3 and its positive feedback regulation through formin. Concurrently, Gli3 functions independently of formin during initial posterior nesting of 5'HoxD domains, whereas their subsequent distal restriction and anterior expansion depends on genetic interaction of Gli3 and formin.

Animals↗
PubMed2004

Formin homology 2 domains occur in multiple contexts in angiosperms.

BACKGROUND: Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. RESULTS: In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. CONCLUSIONS: The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity.

Actins↗
PubMed2005

Rho GTPase-formin pairs in cytoskeletal remodelling.

Diaphanous-related formins (Drfs) are members of a conserved formin family of actin-nucleating proteins and are thought to act as Rho GTPase effectors in signal transduction pathways that govern gene expression, cytoskeletal remodelling and cell division. In vitro evidence suggests that the three mammalian Drf proteins--mDia1, mDia2 and mDia3-have distinct GTPase-binding specificities. However, much of our current understanding of GTPase-Drf partnerships in mammalian cell signalling is based on expression studies using Drfs missing their unique GTPase-binding domains. We have employed fluorescence resonance energy transfer (FRET) and gene targeting approaches to identify the function of different GTPase-formin pairs in cell signalling. These studies have allowed us to uncover new roles for Drf proteins in cytoskeletal remodelling and novel regulatory mechanisms whereby GTPases influence formin function. Our genetic experiments strongly suggest that Drfs cooperate with other GTPase effector proteins, including the gene product of the Wiskott-Aldrich syndrome gene, WASP, during the regulation of cell proliferation. Further, the Drf gene knockout experiments indicate that this family of formins has a role in cancer pathophysiology.

Actins↗
PubMed2005

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.

Actin Cytoskeleton↗
PubMed2006

Dynamics of the formin for3p in actin cable assembly.

BACKGROUND: Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. In the fission yeast Schizosaccharomyces pombe, the formin for3p is necessary for the formation of actin cables, which are bundles of short parallel actin filaments that regulate cell polarity. These filaments are largely organized with their barbed ends facing the cell tip, where for3p is thought to function in their assembly. RESULTS: Here, using a functional for3p-3GFP fusion expressed at endogenous levels, we find that for3p localizes to small dots that appear transiently at cell tips and then move away on actin cables at a rate of 0.3 microm/s. These movements were dependent on the continuous assembly of actin in cables, on the ability of for3p to bind actin within its FH2 domain, and on profilin and bud6p, two formin binding proteins that promote formin activity. Bud6p transiently colocalizes with for3p at the cell tip and stays behind at the cell tip when for3p detaches. CONCLUSIONS: These findings suggest a new model for actin cable assembly: a for3p particle is activated and promotes the assembly of a short actin filament at the cell tip for only seconds. For3p and the actin filament may then be released from the cell tip and carried passively into the cell interior by retrograde flow of actin filaments in the cable. These studies reveal a complex and dynamic cycle of formin regulation and actin cable assembly in vivo.

Actin Cytoskeleton↗
PubMed2006

A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin.

Actin-filament bundles (or cables) have a structural role during cell division and morphogenesis, but also serve as important "tracks" for the transport of materials during cytokinesis and polarized cell growth. However, the dynamic formation of these longitudinal actin-filament higher-order structures is not understood. Recently, several lines of evidence suggest that formins provide one avenue for the initiation of actin cables in vivo. A popular model for the mechanism of polymerization of actin filaments by formin involves the processive movement of formin attached at the barbed end of an elongating filament. In the present study, we use an in vitro system to reconstitute the dynamic formation of actin-filament bundles generated by Arabidopsis FORMIN1 (AFH1). To be able to visualize individual events in such a complex system, we used real-time evanescent-wave microscopy. Surprisingly, we find that AFH1 is a nonprocessive formin that moves from the barbed end to the side of an actin filament after the nucleation event. We show why this new mechanism of nucleation by a member of the formin family is important for bundle formation. Finally, we analyze the different parameters controlling the dynamic formation of such longitudinal actin-filament bundles.

Actin Cytoskeleton↗
PubMed2006

Model of formin-associated actin filament elongation.

Formin FH2 domains associate processively with actin-filament barbed ends and modify their rate of growth. We modeled how the elongation rate depends on the concentrations of profilin and actin for four different formins. We assume that (1) FH2 domains are in rapid equilibrium among conformations that block or allow actin addition and that (2) profilin-actin is transferred rapidly to the barbed end from multiple profilin binding sites in formin FH1 domains. In agreement with previous experiments discussed below, we find an optimal profilin concentration with a maximal elongation rate that can exceed the rate of actin alone. High profilin concentrations suppress elongation, largely because free profilin displaces profilin-actin from FH1. The model supports a common polymerization mechanism for the four formin FH1FH2 constructs with differences attributed to varying parameter values. The mechanism does not require ATP hydrolysis by polymerized actin, but we cannot exclude that formins accelerate hydrolysis.

Actins↗
PubMed2007

How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity.

Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.

Actins↗
PubMed2003

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin.

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.

Actin Cytoskeleton↗
PubMed2003

Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane.

Formins, actin-nucleating proteins that stimulate the de novo polymerization of actin filaments, are important for diverse cellular and developmental processes, especially those dependent on polarity establishment. A subset of plant formins, referred to as group I, is distinct from formins from other species in having evolved a unique N-terminal structure with a signal peptide, a Pro-rich, potentially glycosylated extracellular domain, and a transmembrane domain. We show here that overexpression of the Arabidopsis formin AFH1 in pollen tubes induces the formation of arrays of actin cables that project into the cytoplasm from the cell membrane and that its N-terminal structure targets AFH1 to the cell membrane. Pollen tube elongation is a polar cell growth process dependent on an active and tightly regulated actin cytoskeleton. Slight increases in AFH1 stimulate growth, but its overexpression induces tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. These results suggest that AFH1-regulated actin polymerization is important for the polar pollen cell growth process. Moreover, severe membrane deformation was observed in the apical region of tip-expanded, AFH1-overexpressing pollen tubes in which an abundance of AFH1-induced membrane-associated actin cables was evident. These observations suggest that regulated AFH1 activity at the cell surface is important for maintaining tip-focused cell membrane expansion for the polar extension of pollen tubes. The cell surface-located group-I formins may play the integrin-analogous role as mediators of external stimuli to the actin cytoskeleton, and AFH1 could be important for mediating extracellular signals from female tissues to elicit the proper pollen tube growth response during pollination.

Actins↗
PubMed2005

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.

Actins↗