The Ability to Induce Microtubule Acetylation Is a General Feature of Formin Proteins

Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.     

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.     

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.     

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.     

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.     

Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.     

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.     

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.     

Expression of GFP-mTalin reveals an actin-related role for the Arabidopsis Class II formin AtFH12

Formins (FH2 proteins) are implicated in F-actin nucleation and other aspects of cytoskeletal organization. Plants possess two formin clades, relatively well-described Class I formins and so far poorly characterized Class II formins. Comparison of Class II formin genes of two Arabidopsis species, A. thaliana and A. lyrata, indicates dynamic evolution within the Class II formin clade. Disruption of an outlier A. thaliana Class II formin gene, AtFH12 (At1g42980), whose expression is induced by NaCl, produced only negligible phenotypic effects under a variety of conditions, including salt stress, suggesting functional redundancy among Class II formins. However, the same mutation massively aggravated toxic effects of the expression of a fluorescent actin marker, GFP-tagged mouse talin (GFP-mTalin), known to interfere with normal actin dynamics. Abnormal actin structures were observed in atfh12 mutants expressing GFP-mTalin as compared to wild type. This not only demonstrates an actin-associated function for AtFH12, but also documents the feasibility of using the heterologous actin marker to “stress-test” the actin cytoskeleton in phenotyping “weak“ actin related mutant alleles.     

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.     

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.     

Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1-5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.     

Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies

Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.     

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.     

Plant formins: diverse isoforms and unique molecular mechanism

The completed genome from the model plant Arabidopsis thaliana reveals the presence of a diverse multigene family of formin-like sequences, comprising more than 20 isoforms. This review highlights recent findings from biochemical, cell biological and reverse-genetic analyses of this family of actin nucleation factors. Important advances in understanding cellular function suggest major roles for plant formins during cytokinesis and cell expansion. Biochemical studies on a subset of plant formins emphasize the need to examine molecular mechanisms outside of mammalian and yeast systems. Notably, a combination of solution-based assays for actin dynamics and timelapse, single-filament imaging with TIRFM provide evidence for the first non-processive formin (AtFH1) in eukaryotes. Despite these advances it remains difficult to generate a consensus view of plant formin activities and cellular functions. One limitation to summarizing formin properties relates to the enormous variability in domain organization among the plant formins. Generating homology-based predictions that depend on conserved domains outside of the FH1 and FH2 will be virtually impossible for plant formins. A second major drawback is the lack of facile techniques for examining dynamics of individual actin filaments within live plant cells. This constraint makes it extremely difficult to bridge the gap between biochemical characterization of particular formin and its specific cellular function. There is promise, however, that recent technical advances in engineering appropriate fluorescent markers and new fluoresence imaging techniques will soon allow the direct visualization of cortical actin filament dynamics. The emergence of other model systems for studying actin cytoskeleton in vivo, such as the moss Physcomitrella patens, may also enhance our knowledge of plant formins.     

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.     

Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.     

New insights into the role of plant formins: regulating the organization of the actin and microtubule cytoskeleton

Formins are well-known as important regulators participating in the organization of the actin cytoskeleton in organisms. For many years in the past, research on plant formins is more difficult than that in other eukaryotic formins and is limited to class I formins. Nevertheless, positive progress has been made in plant formin research recently, especially the investigations on class II formins. New functions of plant formins are identified gradually, such as regulating cell division and affecting diffuse cell expansion. More significantly, plant formins are also verified to interact with microtubules in vivo and in vitro. They may probably function as linking proteins between microtubules and microfilaments to participate in various cellular processes.     

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.