Barbed Ends Of Actin Filaments Research Articles

Processive capping by formin suggests a force-driven mechanism of actin polymerization

Regulation of actin polymerization is essential for cell functioning. Here, we predict a novel phenomenon—the force-driven polymerization of actin filaments mediated by proteins of the formin family. Formins localize to the barbed ends of actin filaments, but, in contrast to the standard capping proteins, allow for actin polymerization in the barbed direction. First, we show that the mechanism of such “leaky capping” can be understood in terms of the elasticity of the formin molecules. Second, we demonstrate that if a pulling force acts on the filament end via the leaky cap, the elastic stresses can drive actin polymerization. We estimate that a moderate pulling force of ∼3.4 pN is sufficient to reduce the critical actin concentration required for barbed end polymerization by an order of magnitude. Furthermore, the pulling force increases the polymerization rate. The suggested mechanism of force-driven polymerization could be a key element in a variety of cellular mechanosensing devices.

There goes the neighbourhood: Eps8 joins the barbed-end crowd

The finding that Eps8 caps the barbed ends of actin filaments adds to the growing list of factors responsible for controlling the dynamics of actin polymerization. The requirements for these capping proteins may vary in each cellular context, but EPS-8 seems to be critical during intestinal morphogenesis in the worm.

Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.

Profilin for processivity

![][1] Profilin helps formin to steer actin monomers into the barbed ends of actin filaments. CARLIER/ELSEVIER Profilin helps formin to speed actin assembly and enhance ATP hydrolysis, according to Stephane Romero, Marie-France Carlier (CNRS, Gif-sur-Yvette, France), and colleagues

Two billion years of actin. Meeting on cytoskeletal dynamics: from cell biology to developmental disease.

This meeting began with personal retrospective presentations from two of the founding fathers of European cytoskeletal research—V. Small (Vienna, Austria) and U. Lindberg (Stockholm, Sweden)—to commemorate the thirtieth anniversary of the first definitive publication to establish the existence of an actin cytoskeleton in non‐muscle cells (Lazarides & Weber, 1974). ### Ancient cytoskeletons Whereas other meetings often feature an occasional talk on the prokaryotic cytoskeleton, this meeting devoted a whole session to several aspects of our present knowledge of these ancient systems. This was well received and served as a useful reminder that the cytoskeleton is not just the domain of eukaryotic cell biologists. J. Errington (Oxford, UK) introduced the topic of the bacterial cytoskeleton and reviewed earlier studies that showed the filamentous nature of the ancient bacterial actin proteins MreB and Mbl (MreB‐like). As he pointed out, although we have known about actin filaments for 30 years, they have almost certainly existed for more than two billion years. Many early studies on bacterial cell shape indicated that the cell wall alone conveyed the information that was required for morphology. However, the finding that some of the genes that had been identified in mutational cell‐shape screens encoded protein products that resided in the cytosol meant that this idea had to be revisited. Errington and colleagues are now investigating the functions of the Mbl protein, which is one of three MreB homologues in Bacillus subtilis . They have used fluorescence recovery after photobleaching (FRAP), which is a technically demanding technique in an organism as small as a bacterium, to show that the helical Mbl cables are dynamic structures in vivo (Fig 1). The kinetics of recovery indicate that this is achieved through turnover from unbleached parts of the cell. These studies also imply that the organization of the cables is not likely to be …

Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces.

Formins are large multidomain proteins required for assembly of actin cables that contribute to the polarity and division of animal and fungal cells. Formin homology-1 (FH1) domains bind profilin, and highly conserved FH2 domains nucleate actin filaments. We characterized the effects of two formins, budding yeast Bni1p and fission yeast Cdc12p, on actin assembly. We used evanescent wave fluorescence microscopy to observe assembly of actin filaments (i) nucleated by soluble formin FH1FH2 domains and (ii) associated with formin FH1FH2 domains immobilized on microscope slides. Bni1p(FH1FH2)p and Cdc12p(FH1FH2)p nucleated new actin filaments or captured the barbed ends of preformed actin filaments that grew by insertion of subunits between the immobilized formin and the barbed end of the filament. Both formins remained bound to growing actin filament barbed ends for >1,000 sec. Elongation of a filament between an immobilized formin and a second anchor point buckled filament segments as short as 0.7 microm, demonstrating that polymerization of single actin filaments produces forces of >1 piconewton, close to the theoretical maximum. After buckling, further growth produced long loops that did not supercoil, suggesting that formins do not stair step along the two subunits exposed on the growing barbed end. In agreement, Arp2/3 complex branched filaments did not rotate as they grew from formins attached to the slide surface. Formins are not mechanistically identical because barbed end elongation from Cdc12(FH1FH2)p, but not Bni1(FH1FH2)p, requires profilin. However, profilin increased the rate of Bni1(FH1FH2)p-mediated barbed end elongation from 75% to 100% of full-speed.

Lamellipodial Versus Filopodial Mode of the Actin Nanomachinery: Pivotal Role of the Filament Barbed End

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell “chooses” among these alternative modes of “molecular hardware,” we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.

Non-muscle actin filament elongation from complexes of profilin with nucleotide-free actin and divalent cation-free ATP-actin.

Using vertebrate cytoplasmic actin consisting of a mixture of beta and gamma isoforms, we previously characterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry 39, 13176-13188) and F-actin barbed end elongation from profilin-actin (PA) (Kinosian, H. J., et al. (2002) Biochemistry 41, 6734-6743). Our initial calculations indicated that elongation of F-actin from PA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, the overall actin elongation reaction scheme described by these two linked reactions appeared to be thermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATP binding by actin reduces the negative free energy change for the formation of profilin-MgATP-actin from MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to be thermodynamically balanced. In our present work, we test this hypothesis by measuring actin filament barbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). We find that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation of F-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bound divalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experiments with divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reaction scheme. Thus, the data in this present report support our earlier hypothesis.

The Mouse Formin, FRLα, 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.

Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance.

The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.

F-actin Capping (CapZ) and Other Contractile Saphenous Vein Smooth Muscle Proteins Are Altered by Hemodynamic Stress: A PROTEOMIC APPROACH

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin.

Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.

Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle.

Mutations of the gene encoding unconventional myosin XVa are associated with sensorineural deafness in humans (DFNB3) and shaker (Myo15sh2) mice. In deaf Myo15sh2/sh2 mice, stereocilia are short, nearly equal in length, and lack myosin XVa immunoreactivity. We previously reported that myosin XVa mRNA and protein are expressed in cochlear hair cells. We now show that in the mouse, rat, and guinea pig, endogenous myosin XVa localizes to the tips of the stereocilia of the cochlear and vestibular hair cells. Myosin XVa localization overlaps with the barbed ends of actin filaments and extends to the apical plasma membrane of the stereocilia. Gene gun-mediated transfection of mouse inner ear sensory epithelia explants shows selective accumulation of myosin XVa-GFP at the tips of stereocilia, confirming the localization of native myosin XVa. Expression in COS7 cells also reveals targeting of myosin XVa-GFP to the dynamic actin region at the tips of filopodia. In a wild-type mouse, during auditory and vestibular hair cell development, myosin XVa appears at the tips of stereocilia at the time when the hair bundle begins to develop its characteristic staircase pattern. We propose that myosin XVa is essential for the graded elongation of stereocilia during their functional maturation.

Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration.

Ena/VASP proteins are a conserved family of actin regulatory proteins made up of EVH1, EVH2 domains, and a proline-rich central region. They have been implicated in actin-based processes such as fibroblast migration, axon guidance, and T cell polarization and are important for the actin-based motility of the intracellular pathogen Listeria monocytogenes. Mechanistically, these proteins associate with barbed ends of actin filaments and antagonize filament capping by capping protein (CapZ). In addition, they reduce the density of Arp2/3-dependent actin filament branches and bind Profilin at sites of actin polymerization. Vertebrate Ena/VASP proteins are substrates for PKA/PKG serine/threonine kinases. Phosphorylation by these kinases appears to modulate Ena/VASP function within cells, although the mechanism underlying this regulation remains to be determined.

Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actin-binding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with Kd values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.

How VASP enhances actin-based motility.

The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

Depolymerization of Actin Filaments by Profilin: EFFECTS OF PROFILIN ON CAPPING PROTEIN FUNCTION

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.

Formins: signaling effectors for assembly and polarization of actin filaments.

Eukaryotic cells require filamentous actin to maintain their shape and for movement, growth and replication. New actin filaments are formed by the cutting of existing filaments or de novo through the action of specialized nucleators. The most highly characterized nucleator is the Arp2/3 complex, which nucleates the branched actin networks in the lamellae of migrating cells. Recently, Bni1p, which is a member of the formin family of proteins, has been shown to nucleate actin filaments in vitro. Formins are implicated in the formation of actin cables in yeast, stress fibers in tissue culture cells and cytokinesis in many cell types. Formins contain two highly conserved formin-homology domains, FH1 and FH2. The Bni1p FH2 domain is sufficient to mediate nucleation. The Bni1p FH1 domain binds profilin, an actin-monomer-binding protein that delivers actin to the growing barbed end of filaments. The Bni1p FH1-profilin interaction enhances nucleation. Formins participate in a number of signaling pathways that control the assembly of specific actin structures and bind the barbed end of actin filaments, thereby providing a cytoskeletal basis for the establishment of cell polarity.

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

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

Role for phosphoinositide 3-kinase in Fc gamma RIIA-induced platelet shape change.

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-Fc gamma RIIA antibody. Fc gamma RIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows Fc gamma RIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by Fc gamma RIIA and clearly distinguish the Fc gamma RIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway.