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Thomas D. Pollard - One of the best experts on this subject based on the ideXlab platform.
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gating mechanisms during actin filament elongation by Formins
eLife, 2018Co-Authors: Fikret Aydin, Naomi Courtemanche, Thomas D. Pollard, Gregory A VothAbstract:Formins play an important role in the polymerization of unbranched actin filaments, and particular Formins slow elongation by 5-95%. We studied the interactions between actin and the FH2 domains of Formins Cdc12, Bni1 and mDia1 to understand the factors underlying their different rates of polymerization. All-atom molecular dynamics simulations revealed two factors that influence actin filament elongation and correlate with the rates of elongation. First, FH2 domains can sterically block the addition of new actin subunits. Second, FH2 domains flatten the helical twist of the terminal actin subunits, making the end less favorable for subunit addition. Coarse-grained simulations over longer time scales support these conclusions. The simulations show that filaments spend time in states that either allow or block elongation. The rate of elongation is a time-average of the degree to which the formin compromises subunit addition rather than the formin-actin complex literally being in 'open' or 'closed' states.
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the role of intermolecular interactions in the polymerization of actin by Formins
Biophysical Journal, 2017Co-Authors: Fikret Aydin, Naomi Courtemanche, Thomas D. Pollard, Gregory A VothAbstract:Formins are large multidomain proteins that nucleate and modulate the elongation of actin filaments. The FH2 domains (∼400 amino acids) remain associated with the growing barbed end and slow elongation by 5 to 95% depending on the particular formin. We use all atom and coarse-grained molecular dynamics simulations to investigate the interactions of three Formins FH2 domains (fission yeast Cdc12, budding yeast Bni1p, mouse mDia1) and the barbed end of the actin filament to understand these Formins polymerize actin at different rates. The simulations demonstrated that dissimilarities in the structures and intermolecular interactions of three formin-actin complexes, which can affect their elongation rates. In addition, the interactions between FH2 domains and actin filament affect the configuration of the barbed end of the filament, and we identified critical regions of Formins that play role in regulating the barbed-end configuration. Our coarse-grained simulations predicted these Formins have different degrees of steric interference to the addition of actin monomer to the barbed end, and it was found to be inversely proportional to their rates of polymerization. This work can help to resolve the underlying molecular mechanisms used by different Formins to assemble actin networks.
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tension modulates actin filament polymerization mediated by formin and profilin
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Naomi Courtemanche, Thomas D. Pollard, Ja Yil Lee, Eric C GreeneAbstract:Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin–actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p.
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Determinants of formin homology 1 (FH1) domain function in actin filament elongation by Formins
Journal of Biological Chemistry, 2012Co-Authors: Naomi Courtemanche, Thomas D. PollardAbstract:Formin-mediated elongation of actin filaments proceeds via association of Formin Homology 2 (FH2) domain dimers with the barbed end of the filament, allowing subunit addition while remaining processively attached to the end. The flexible Formin Homology 1 (FH1) domain, located directly N-terminal to the FH2 domain, contains one or more stretches of polyproline that bind the actin-binding protein profilin. Diffusion of FH1 domains brings associated profilin-actin complexes into contact with the FH2-bound barbed end of the filament, thereby enabling direct transfer of actin. We investigated how the organization of the FH1 domain of budding yeast formin Bni1p determines the rates of profilin-actin transfer onto the end of the filament. Each FH1 domain transfers actin to the barbed end independently of the other and structural evidence suggests a preference for actin delivery from each FH1 domain to the closest long-pitch helix of the filament. The transfer reaction is diffusion-limited and influenced by the affinities of the FH1 polyproline tracks for profilin. Position-specific sequence variations optimize the efficiency of FH1-stimulated polymerization by binding profilin weakly near the FH2 domain and binding profilin more strongly farther away. FH1 domains of many other Formins follow this organizational trend. This particular sequence architecture may optimize the efficiency of FH1-stimulated elongation.
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review of the mechanism of processive actin filament elongation by Formins
Cytoskeleton, 2009Co-Authors: Aditya S Paul, Thomas D. PollardAbstract:We review recent structural and biophysical studies of the mechanism of action of Formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addition to variable regulatory domains, Formins typically have formin homology 1 (FH1) and formin homology 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains concentrate multiple profilin-actin complexes near the end of the filament. FH1 domains transfer actin very rapidly onto the barbed end of the filament, allowing elongation at rates that exceed the rate of elongation by the addition of free actin monomers diffusing in solution. Binding of actin to the end of the filament provides the energy for the highly processive movement of the FH2 as a filament adds thousands of actin subunits. These biophysical insights provide the context to understand how Formins contribute to actin assembly in cells. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
Henry N Higgs - One of the best experts on this subject based on the ideXlab platform.
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mutations to the formin homology 2 domain of inf2 protein have unexpected effects on actin polymerization and severing
Journal of Biological Chemistry, 2012Co-Authors: Vinay Ramabhadran, Pinar S Gurel, Henry N HiggsAbstract:INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other Formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other Formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all Formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.
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Differential interactions of the Formins INF2, mDia1, and mDia2 with microtubules.
Molecular Biology of the Cell, 2011Co-Authors: Jeremie Gaillard, Vinay Ramabhadran, Emmanuelle Neumann, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N HiggsAbstract: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.
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Rho activation of mDia Formins is modulated by an interaction with inverted formin 2 (INF2)
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Hua Sun, Henry N Higgs, Johannes Schlondorff, Elizabeth J. Brown, Martin R. PollakAbstract:Inverted formin 2 (INF2) encodes a member of the diaphanous subfamily of formin proteins. Mutations in INF2 cause human kidney disease characterized by focal and segmental glomerulosclerosis. Disease-causing mutations occur only in the diaphanous inhibitory domain (DID), suggesting specific roles for this domain in the pathogenesis of disease. In a yeast two-hybrid screen, we identified the diaphanous autoregulatory domains (DADs) of the mammalian diaphanous-related Formins (mDias) mDia1, mDia2, and mDia 3 as INF2_DID-interacting partners. The mDias are Rho family effectors that regulate actin dynamics. We confirmed in vitro INF2_DID/mDia_DAD binding by biochemical assays, confirmed the in vivo interaction of these protein domains by coimmunoprecipitation, and observed colocalization of INF2 and mDias in glomerular podocytes. We investigated the influence of this INF2_DID/mDia_DAD interaction on mDia mediated actin polymerization and on serum response factor (SRF) activation. We find that the interaction of INF2_DID with mDia_DAD inhibited mDia-mediated, Rho-activated actin polymerization, as well as SRF-responsive gene transcriptional changes. Similar assays using the disease-causing E184K and R218Q mutations in INF2_DID showed a decreased effect on SRF activation and gene transcription. The binding of INF2_DID to mDia_DAD may serve as a negative regulatory mechanism for mDias’ function in actin-dependent cell processes. The effects of disease-causing INF2 mutations suggest an important role for this protein and its interaction with other Formins in modulating glomerular podocyte phenotype and function.
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the filamentous actin cross linking bundling activity of mammalian Formins
Journal of Molecular Biology, 2008Co-Authors: Osigwe Esue, Henry N Higgs, Elizabeth S Harris, Denis WirtzAbstract:Summary 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 α-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 α-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 α-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 α-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.
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inf2 is a wasp homology 2 motif containing formin that severs actin filaments and accelerates both polymerization and depolymerization
Journal of Biological Chemistry, 2006Co-Authors: Ekta Seth Chhabra, Henry N HiggsAbstract:Abstract 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.
Bruce L Goode - One of the best experts on this subject based on the ideXlab platform.
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critical roles for multiple Formins during cardiac myofibril development and repair
Molecular Biology of the Cell, 2014Co-Authors: Michelle Rosado, Cynthia F Barber, Cristina Berciu, Steven Feldman, Susan J Birren, Daniela Nicastro, Bruce L GoodeAbstract:Cardiac and skeletal muscle function depends on the proper formation of myofibrils, which are tandem arrays of highly organized actomyosin contractile units called sarcomeres. How the architecture of these colossal molecular assemblages is established during development and maintained over the lifetime of an animal is poorly understood. We investigate the potential roles in myofibril formation and repair of formin proteins, which are encoded by 15 different genes in mammals. Using quantitative real-time PCR analysis, we find that 13 Formins are differentially expressed in mouse hearts during postnatal development. Seven Formins immunolocalize to sarcomeres in diverse patterns, suggesting that they have a variety of functional roles. Using RNA interference silencing, we find that the Formins mDia2, DAAM1, FMNL1, and FMNL2 are required nonredundantly for myofibrillogenesis. Knockdown phenotypes include global loss of myofibril organization and defective sarcomeric ultrastructure. Finally, our analysis reveals an unanticipated requirement specifically for FMNL1 and FMNL2 in the repair of damaged myofibrils. Together our data reveal an unexpectedly large number of Formins, with diverse localization patterns and nonredundant roles, functioning in myofibril development and maintenance, and provide the first evidence of actin assembly factors being required to repair myofibrils.
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essential and nonredundant roles for diaphanous Formins in cortical microtubule capture and directed cell migration
Molecular Biology of the Cell, 2014Co-Authors: Pascale Daou, Bruce L Goode, Dennis Breitsprecher, Salma Hasan, Emilie Baudelet, Luc Camoin, Stephane Audebert, Ali BadacheAbstract:Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related Formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three Formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three Formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.
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structure and activity of full length formin mdia1
Cytoskeleton, 2012Co-Authors: Sankar Maiti, Laurent Blanchoin, Alphee Michelot, Christopher J Gould, O S Sokolova, Bruce L GoodeAbstract:Formins are a conserved family of actin assembly-promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on Formins has been limited to fragments. This has left open key questions about the structure, activity and regulation of intact formin proteins. Here, we isolated full-length mouse mDia1 (mDia1-FL) and found that it forms tightly autoinhibited dimers that can only be partially activated by RhoA. We solved the structure of autoinhibited mDia1-FL using electron microscopy and single particle analysis. Docking of crystal structures into the 3D reconstruction revealed that the fork-shaped N-terminal DID-CC region hangs over the ring-shaped FH2 domain, suggesting that autoinhibition results from steric obstruction of actin binding. Deletion of the C-terminal DAD domain extended mDia1 structure and activated it for actin assembly. Using TIRF microscopy, we observed that RhoA-activated mDia1-FL persistently accelerated filament elongation in the presence of profilin similar to mDia1 FH1-FH2 fragment. These observations validate the known activities of FH1-FH2 fragments as reflecting those of the intact molecule. Our results further suggest that mDia1-FL does not readily snap back into the autoinhibited conformation and dissociate from growing filament ends, and thus additional factors may be required to displace Formins and restrict filament length.
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displacement of Formins from growing barbed ends by bud14 is critical for actin cable architecture and function
Developmental Cell, 2009Co-Authors: Melissa A Chesarone, Christopher J Gould, James B Moseley, Bruce L GoodeAbstract:Summary Normal cellular development and function require tight spatiotemporal control of actin assembly. Formins are potent actin assembly factors that protect the growing ends of actin filaments from capping proteins. However, it is unresolved how the duration of formin-mediated actin assembly events is controlled, whether Formins are actively displaced from growing ends, and how filament length is regulated in vivo. Here, we identify Bud14 as a high-affinity inhibitor of the yeast formin Bnr1 that rapidly displaces the Bnr1 FH2 domain from growing barbed ends. Consistent with these activities, bud14Δ cells display fewer actin cables, which are aberrantly long, bent, and latrunculinA resistant, leading to defects in secretory vesicle movement. Moreover, bud14Δ suppressed mutations that cause abnormally numerous and shortened cables, restoring wild-type actin architecture. From these results, we propose that formin displacement factors regulate filament length and are required in vivo to maintain proper actin network architecture and function.
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mechanism and function of Formins in the control of actin assembly
Annual Review of Biochemistry, 2007Co-Authors: Bruce L Goode, Michael J EckAbstract:Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed Formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of Formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
Fatima Cvrčková - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis class ii Formins atfh13 and atfh14 can form heterodimers but exhibit distinct patterns of cellular localization
International Journal of Molecular Sciences, 2020Co-Authors: Eva Kollarova, Anežka Baquero Forero, Lenka Stillerova, Sylva Přerostova, Fatima CvrčkováAbstract:Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm Formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II Formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length Formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization (including co-localization with cytoskeletal structures and the endoplasmic reticulum) was examined using confocal microscopy. While the two Formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay-a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.
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Formins and membranes anchoring cortical actin to the cell wall and beyond
Frontiers in Plant Science, 2013Co-Authors: Fatima CvrčkováAbstract:Formins are evolutionarily conserved eukaryotic proteins participating in actin and microtubule organization. Land plants have three formin clades, with only two – Class I and II – present in angiosperms. Class I Formins are often transmembrane proteins, residing at the plasmalemma and anchoring the cortical cytoskeleton across the membrane to the cell wall, while Class II Formins possess a PTEN-related membrane-binding domain. Lower plant Class III and non-plant Formins usually contain domains predicted to bind RHO GTPases that are membrane-associated. Thus, some kind of membrane anchorage appears to be a common formin feature. Direct interactions between various non-plant Formins and integral or peripheral membrane proteins have indeed been reported, with varying mechanisms and biological implications. Besides of summarizing new data on Class I and Class II formin-membrane relationships, this review surveys such “non-classical” formin-membrane interactions and examines which, if any, of them may be evolutionarily conserved and operating also in plants. FYVE, SH3 and BAR domain-containing proteins emerge as possible candidates for such conserved membrane-associated formin partners.
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Formins: emerging players in the dynamic plant cell cortex.
Scientifica, 2012Co-Authors: Fatima CvrčkováAbstract:Formins (FH2 proteins) are an evolutionarily conserved family of eukaryotic proteins, sharing the common FH2 domain. While they have been, until recently, understood mainly as actin nucleators, Formins are also engaged in various additional aspects of cytoskeletal organization and signaling, including, but not limited to, the crosstalk between the actin and microtubule networks. A surprising diversity of domain organizations has been discovered among the FH2 proteins, and specific domain setups have been found in plants. Seed plants have two clades of Formins, one of them (Class I) containing mostly transmembrane proteins, while members of the other one (Class II) may be anchored to membranes via a putative membrane-binding domain related to the PTEN antioncogene. Thus, plant Formins present good candidates for possible mediators of coordination of the cortical actin and microtubule cytoskeletons, as well as their attachment to the plasma membrane, that is, aspects of cell cortex organization likely to be important for cell and tissue morphogenesis. Although experimental studies of plant formin function are hampered by the large number of formin genes and their functional redundancy, recent experimental work has already resulted in some remarkable insights into the function of FH2 proteins in plants.
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roots of angiosperm Formins the evolutionary history of plant fh2 domain containing proteins
BMC Evolutionary Biology, 2008Co-Authors: Michal Grunt, Viktor žarský, Fatima CvrčkováAbstract: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. The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium Formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I Formins and Class II Formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile Formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) Formins that define a specific branch (Class III) of the formin family. We propose a scenario where Formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II Formins, allowing later loss of the RhoGAP-like domain-containing Formins in angiosperms.
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Roots of angiosperm Formins: The evolutionary history of plant FH2 domain-containing proteins
BMC Evolutionary Biology, 2008Co-Authors: Michal Grunt, Viktor žarský, Fatima CvrčkováAbstract: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. Results The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium Formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I Formins and Class II Formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile Formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) Formins that define a specific branch (Class III) of the formin family. Conclusion We propose a scenario where Formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II Formins, allowing later loss of the RhoGAP-like domain-containing Formins in angiosperms.
Naomi Courtemanche - One of the best experts on this subject based on the ideXlab platform.
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dissection of two parallel pathways for formin mediated actin filament elongation
Journal of Biological Chemistry, 2018Co-Authors: Laura A Sherer, Mark E Zweifel, Naomi CourtemancheAbstract:Formins direct the elongation of unbranched actin filaments that are incorporated into a diverse set of cytoskeletal structures. Elongation of formin-bound filaments occurs along two parallel pathways. The formin homology 2 (FH2) pathway allows actin monomers to bind directly to barbed ends bound by dimeric FH2 domains. The formin homology 1 (FH1) pathway involves transfer of profilin-bound actin to the barbed end from polyproline tracts located in the disordered FH1 domains. Here, we used a total internal reflection fluorescence (TIRF) microscopy-based fluorescence approach to determine the fraction of actin subunits incorporated via the FH1 and FH2 pathways during filament elongation mediated by two Formins. We found that the fraction of filament elongation that occurs via each pathway directly depends on the efficiency of the other pathway, indicating that these two pathways compete with each other for subunit addition by Formins. We conclude that this competition allows Formins to compensate for changes in the efficiency of one pathway by adjusting the frequency of subunit addition via the other, thus increasing the overall robustness of formin-mediated actin polymerization.
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gating mechanisms during actin filament elongation by Formins
eLife, 2018Co-Authors: Fikret Aydin, Naomi Courtemanche, Thomas D. Pollard, Gregory A VothAbstract:Formins play an important role in the polymerization of unbranched actin filaments, and particular Formins slow elongation by 5-95%. We studied the interactions between actin and the FH2 domains of Formins Cdc12, Bni1 and mDia1 to understand the factors underlying their different rates of polymerization. All-atom molecular dynamics simulations revealed two factors that influence actin filament elongation and correlate with the rates of elongation. First, FH2 domains can sterically block the addition of new actin subunits. Second, FH2 domains flatten the helical twist of the terminal actin subunits, making the end less favorable for subunit addition. Coarse-grained simulations over longer time scales support these conclusions. The simulations show that filaments spend time in states that either allow or block elongation. The rate of elongation is a time-average of the degree to which the formin compromises subunit addition rather than the formin-actin complex literally being in 'open' or 'closed' states.
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the role of intermolecular interactions in the polymerization of actin by Formins
Biophysical Journal, 2017Co-Authors: Fikret Aydin, Naomi Courtemanche, Thomas D. Pollard, Gregory A VothAbstract:Formins are large multidomain proteins that nucleate and modulate the elongation of actin filaments. The FH2 domains (∼400 amino acids) remain associated with the growing barbed end and slow elongation by 5 to 95% depending on the particular formin. We use all atom and coarse-grained molecular dynamics simulations to investigate the interactions of three Formins FH2 domains (fission yeast Cdc12, budding yeast Bni1p, mouse mDia1) and the barbed end of the actin filament to understand these Formins polymerize actin at different rates. The simulations demonstrated that dissimilarities in the structures and intermolecular interactions of three formin-actin complexes, which can affect their elongation rates. In addition, the interactions between FH2 domains and actin filament affect the configuration of the barbed end of the filament, and we identified critical regions of Formins that play role in regulating the barbed-end configuration. Our coarse-grained simulations predicted these Formins have different degrees of steric interference to the addition of actin monomer to the barbed end, and it was found to be inversely proportional to their rates of polymerization. This work can help to resolve the underlying molecular mechanisms used by different Formins to assemble actin networks.
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tension modulates actin filament polymerization mediated by formin and profilin
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Naomi Courtemanche, Thomas D. Pollard, Ja Yil Lee, Eric C GreeneAbstract:Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin–actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p.
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Determinants of formin homology 1 (FH1) domain function in actin filament elongation by Formins
Journal of Biological Chemistry, 2012Co-Authors: Naomi Courtemanche, Thomas D. PollardAbstract:Formin-mediated elongation of actin filaments proceeds via association of Formin Homology 2 (FH2) domain dimers with the barbed end of the filament, allowing subunit addition while remaining processively attached to the end. The flexible Formin Homology 1 (FH1) domain, located directly N-terminal to the FH2 domain, contains one or more stretches of polyproline that bind the actin-binding protein profilin. Diffusion of FH1 domains brings associated profilin-actin complexes into contact with the FH2-bound barbed end of the filament, thereby enabling direct transfer of actin. We investigated how the organization of the FH1 domain of budding yeast formin Bni1p determines the rates of profilin-actin transfer onto the end of the filament. Each FH1 domain transfers actin to the barbed end independently of the other and structural evidence suggests a preference for actin delivery from each FH1 domain to the closest long-pitch helix of the filament. The transfer reaction is diffusion-limited and influenced by the affinities of the FH1 polyproline tracks for profilin. Position-specific sequence variations optimize the efficiency of FH1-stimulated polymerization by binding profilin weakly near the FH2 domain and binding profilin more strongly farther away. FH1 domains of many other Formins follow this organizational trend. This particular sequence architecture may optimize the efficiency of FH1-stimulated elongation.