Actin Filament Elongation Research Articles

FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers

Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

1P139 アクチンフィラメントの伸長メカニズムを解明するための分子シミュレーション(10.筋肉,ポスター,日本生物物理学会年会第51回(2013年度))

1P139 アクチンフィラメントの伸長メカニズムを解明するための分子シミュレーション(10.筋肉,ポスター,日本生物物理学会年会第51回(2013年度))

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing

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.

Determinants of Formin Homology 1 (FH1) Domain Function in Actin Filament Elongation by Formins

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.

Structural and Biochemical Study of the Interaction Between Actin and the Mammalian Formin FMNL3

Formins are a class of proteins that influence the rate of actin filament nucleation and elongation. Mammals possess 15 formin isoforms, providing a myriad of possibilities for regulating actin-based structures in cells. The dimeric formin homology 2 (FH2) domain is capable of altering the nucleation rate of new actin filaments and subsequently influences filament elongation via direct interaction with the barbed end of an actin filament. The FH2 domain moves processively with the barbed end as the filament elongates. Our goal in this study is to examine FMNL3's interaction with actin to uncover mechanistic details of formin regulated actin elongation. The FH2 domain of FMNL3 that forms a stable interaction with tetramethylrhodamine-malamide labeled actin (TMR-actin) and has been crystallized and the 3.3 A resolution structure has been solved. The crystal structure indicates that FMNL3 dimerizes to form a ring around the barbed end of two actin subunits. The formin/actin interface is comprised of multiple interactions that are found in three distinct regions of the FH2 domain. Mutational analysis identified key residues in FMNL3/actin binding as well as those essential for elongation activity. The study of these residues has led us to propose a more detailed model for the interaction of the FH2 domain with actin. A previous study with Bni1p, revealed an FH2 dimer conformation spanning three actin subunits. This structure supports the model of a formin tracking with the growing end of an actin filament by taking steps with the addition of each monomer. The FMNL3/actin complex represents an intermediate step in the formin stepping mechanism in which two actin subunits are aligned side-by-side surrounded by a symmetric FH2 dimer. This structure reveals mechanistic details of FH2 mediated actin filament elongation via processive capping.

Nuclear Localization of mDia2 Formin and its Possible Regulation by Rho Family GTPases

Formin family proteins are potent activators of actin filament nucleation and elongation. While numerous studies indicate the possible roles of actin inside the nucleus and the tight association of the nucleus with cytoplasmic actin cytoskeleton, the role of actin-associated proteins and, in particular, regulators of actin polymerization in the organization of nuclear and perinuclear actin remains obscure. Nuclear localization of some formins, such as formin-1 and mDia2 was detected in some previous studies. Particularly, it was recently shown that mDia2 has both nuclear localization and nuclear export signals, and is accumulated in the nucleus upon inhibition of nuclear export by leptomycin B (LMB) (Miki et al, J Biol Chem. 2009). We confirmed and extended these results; in particular, we have measured the kinetics of nuclear accumulation of some formins upon LMB treatment and have found that mDia2 accumulates in the nucleus very fast while mDia1 does not accumulate. Since mDia2 activity is regulated by the Rho family GTPases, we further studied the nuclear localization of RhoA, Rac1, and Cdc42 and found that significant amounts of the wild type Rho GTPases were localized to the nucleus, while constitutively active Rho GTPases demonstrated essentially cytoplasmic localization. Moreover, active Cdc42 was shown to augment localization of mDia2 to cell plasma membrane and slow down its nuclear shuttling. Finally, we have found that even without LMB treatment, GFP-fused full length mDia2 is accumulated to the nuclear rim, where it colocalizes with nesprin, a protein involved in linking nucleus with actin cytoskeleton. Constitutively active Rac1 was also enriched in the nuclear rim. The experiments using cell treatment with digitonin, which permeabilizes plasma- but not the nuclear membrane, suggested that mDia2-enriched nuclear rim was located at the cytoplasmic side of the nuclear membrane.

Numerical Analysis of Intracellular Actin Network Dynamics Related on Cell Movements

Actin dynamics is one of the essential mechanisms for cell movements. Recent studies revealed various proteins required to control actin dynamics in cells, i.e. G-actin for actin assembly and disassembly, Arp2/3 for branching of actin filament, ADF/cofilin for severing of actin filament, capping protein for terminating actin filament elongation and so on. Based on those proteins’ regulatory control, dendritic actin networks would be formed close to the edge of the cell, which leads to the cell protrution, that is the first step of the cell movements. However, because of the difficulty in measuring the actin network dynamics in living cell, it is still unclear how the actin network is remodeled dynamically inside the cell. Therefore, in this study, we developed a computational stochastic model based on chemical reaction equations for the dendritic nucleation model of the actin dynamics using Gillespie's algorithm. In the analysis, local concentration of proteins, rate constant of chemical reaction equations and mechanical relationship between the actin network inside the cell and cell membrane were taken into consideration, and the mechanism of the cell protrution was analyzed.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Barriers to Diabetes Prevention and Control Among American Indians

North Carolina's American Indian population experiences a disproportionate diabetes burden, in terms of both a high prevalence of the disease and excess diabetes-related death and disability. Concerted efforts need to be made to provide culturally appropriate and easily accessible education, health care, and health-promoting resources in these vulnerable communities.

The Myosin Passenger Protein Smy1 Controls Actin Cable Structure and Dynamics by Acting as a Formin Damper

Formins are a conserved family of proteins with robust effects in promoting actin nucleation and elongation. However, the mechanisms restraining formin activities in cells to generate actin networks with particular dynamics and architectures are not well understood. In S.cerevisiae, formins assemble actin cables, which serve as tracks for myosin-dependent intracellular transport. Here, we show that the kinesin-like myosin passenger-protein Smy1 interacts with the FH2 domain of the formin Bnr1 to decrease rates of actin filament elongation, which is distinct from the formin displacement activity of Bud14. Invivo analysis of smy1Δ mutants demonstrates that this "damper" mechanism is critical for maintaining proper actin cable architecture, dynamics, and function. We directly observe Smy1-3GFP being transported by myosin V and transiently pausing at the neck in a manner dependent on Bnr1. These observations suggest that Smy1 is part of a negative feedback mechanism that detects cable length and prevents overgrowth.

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

Molecular mechanism of Ena/VASP-mediated actin-filament elongation

Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G-actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP-mediated filament elongation depends on G-actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP-mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP-mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.

The Mechanics of Movement

Within cells, formin proteins promote the elongation of cytoskeletal actin filaments by associating with filament tips. This activity has the potential to harness actin-generated pushing forces to change cellular architecture. Mizuno et al. (p. [80][1], published online 9 December; see the Perspective by [Pollard][2] ) have devised a simple method to analyze the movement of single molecules of a member of the formin family, mDia1, along the growing actin filaments; they discovered that formin molecules mechanically rotate on the end of actin filaments, both during growth and depolymerization of the filament, and can thus influence cell shape during important stages in the cell cycle. [1]: /lookup/doi/10.1126/science.1197692 [2]: /lookup/doi/10.1126/science.1200773

Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance

Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle-specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification.

Assembly of filopodia by the formin FRL2 (FMNL3)

Actin-dependent finger-like protrusions such as filopodia and microvilli are widespread in eukaryotes, but their assembly mechanisms are poorly understood. Filopodia assembly requires at least three biochemical activities on actin: actin filament nucleation, prolonged actin filament elongation, and actin filament bundling. These activities are shared by several mammalian formin proteins, including mDia2, FRL1 (also called FMNL1), and FRL2 (FMNL3). In this paper, we compare the abilities of constructs from these three formins to induce filopodia. FH1-FH2 constructs of both FRL2 and mDia2 stimulate potent filopodia assembly in multiple cell types, and enrich strongly at filopodia tips. In contrast, FRL1 FH1-FH2 lacks this activity, despite possessing similar biochemical activities and being highly homologous to FRL2. Chimeric FH1-FH2 experiments between FRL1 and FRL2 show that, while both an FH1 and an FH2 are needed, either FH1 domain supports filopodia assembly but only FRL2's FH2 domain allows this activity. A mutation that compromises FRL2's barbed end binding ability abolishes filopodia assembly. FRL2's ability to stimulate filopodia assembly is not altered by additional domains (GBD, DID, DAD), but is significantly reduced in the full-length construct, suggesting that FRL2 is subject to inhibitory regulation. The data suggest that the FH2 domain of FRL2 possesses properties not shared by FRL1 that allow it to generate filopodia.

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.

Direct dynamin–actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

A mathematical model of the sarcomere mechanics of cross-striated muscle with consideration of the stretch and twist of actin filaments

A mathematical model of sarcomere mechanics, which takes into account the elongation of actin and myosin filaments and also twisting of the actin filaments in the sarcomere of striated muscle during contraction is presented. The model accounts for the experimentally observed phenomena of the stretch and twist of the actin filaments due to strong binding of myosin heads and the pulling force. Some model parameters were estimated from published experimental data. The results of modeling show that the twist of actin filaments can play a substantial role in the mechanical responses of contracting muscle fibers to step changes of their length.

LIM kinase mediates estrogen action on the actin depolymerization factor Cofilin

The ovarian hormone estrogen increases the axospinous synapse density in the hippocampal CA1 region of young female rats but fails to do so in aged rats. This estrogen-mediated alteration of spine synapse structures suggests the coincident requirement for the structural reorganization of the underlying actin cytoskeleton network. Actin reorganization is known to require the deactivation of Cofilin, an actin depolymerization factor. Cofilin is deactivated by LIM kinase (LIMK), and LIMK activity is modulated by the phosphorylation of specific residues. We have previously demonstrated that estrogen is able to increase phosphorylated LIMK (pLIMK) immunoreactivity (IR) in the hippocampus in vivo and that this estrogen-stimulated pLIMK-IR is decreased in the aged brain. Because Cofilin phosphorylation allows for actin filament elongation and spine synapse growth, we sought to determine if estrogen acts through Cofilin and if such estrogen action requires the observed LIMK activity. Using both hippocampal neurons and the NG108-15 neuroblastoma cell line, we demonstrate here that estrogen stimulates the phosphorylation of Cofilin in vitro. Furthermore, this estrogen action on Cofilin requires LIMK. Lastly, while initiating the phosphorylation of LIMK and Cofilin, estrogen can also stimulate the formation of filopodial extensions, an early step in the formation of nascent spines, demonstrating that estrogen can alter the actin-dependent neuronal morphology. This linkage of estrogen communication to Cofilin via LIMK provides the functionality to the age-sensitive pLIMK-IR that we have observed in vivo.