Actin Filament Elongation Research Articles

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.

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

Tropomodulins (Tmod) bind to the N terminus of tropomyosin and cap the pointed end of actin filaments. Tropomyosin alone also inhibits the rate of actin depolymerization at the pointed end of filaments. Here we have defined 1) the structural requirements of the N terminus of tropomyosin important for regulating the pointed end alone and with erythrocyte Tmod (Tmod1), and 2) the Tmod1 subdomains required for binding to tropomyosin and for regulating the pointed end. Changes in pyrene-actin fluorescence during polymerization and depolymerization were measured with actin filaments blocked at the barbed end with gelsolin. Three tropomyosin isoforms differently influence pointed end dynamics. Recombinant TM5a, a short non-muscle alpha-tropomyosin, inhibited depolymerization. Recombinant (unacetylated) TM2 and N-acetylated striated muscle TM (stTM), long alpha-tropomyosin isoforms with the same N-terminal sequence, different from TM5a, also inhibited depolymerization but were less effective than TM5a. All blocked the pointed end with Tmod1 in the order of effectiveness TM5a >stTM >TM2, showing the importance of the N-terminal sequence and modification. Tmod1-(1-344), lacking the C-terminal 15 residues, did not nucleate polymerization but blocked the pointed end with all three tropomyosin isoforms as does a shorter fragment, Tmod1-(1-92), lacking the C-terminal "capping" domain though higher concentrations were required. An even shorter fragment, Tmod1-(1-48), bound tropomyosin but did not influence actin filament elongation. Tropomyosin-Tmod may function to locally regulate cytoskeletal dynamics in cells by stabilizing actin filaments.

ENA/VASP proteins: multifunctional regulators of actin cytoskeleton dynamics.

The spatial and temporal regulation of the actin cytoskeleton is fundamental to several cellular processes as diverse as cell motility and immune responses. At the molecular level, the remodelling of the actin cytoskeleton depends on two key events: actin filament nucleation and elongation. Seminal studies on the actin-based intracellular motility of the bacterial pathogen Listeria monocytogenes have been instrumental for the characterisation of a class of actin filament elongating factors, the proteins of the Ena/VASP family. Ena/VASP proteins enhance actin filament elongation via the recruitment of profilin:actin complexes to sites of active actin remodelling such as the tips of spreading lamellipodia and the surface of intracellular Listeria. Moreover, Ena/VASP proteins not only enhance actin filament elongation but also influence the activity of the Arp2/3 complex and counteract the inhibition of actin polymerisation by capping proteins. These findings, taken together with the observation that Ena/VASP proteins can influence actin filament architecture by affecting the actin filament branching activity of the Arp2/3 complex, define Ena/VASP proteins as multifunctional organisers of the actin cytoskeleton.

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles.

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.

Do filopodia enable the growth cone to find its way?

Ena/VASP family proteins are required for accurate axon pathfinding in developing Drosophila, Caenorhabditis elegans, and mouse nervous systems. Recent papers by Bear et al. and Svitkina et al. provide evidence that Ena/VASP proteins regulate actin filament elongation in the lamellipodia and filopodia of nonneuronal cells. Neuronal growth cones use filopodia as their "sensory organs" that steer the growth cone by reading guidance cues in the environment. Together, these studies suggest that Ena/VASP proteins regulate axon guidance by controlling the formation or rearrangement of growth cone filopodia.

Unconventional myosins, actin dynamics and endocytosis: a ménage à trois?

Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation.

A crucial role for profilin–actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility.

Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization.

The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.

A Cross-linked Profilin-Actin Heterodimer Interferes with Elongation at the Fast-growing End of F-actin

Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.

Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility

Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin.

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.

Intracellular invasion of Staphylococcus aureus against human gingival fibroblasts

Staphylococcus aureus (S. aureus) is one of the most common bacterial pathogens, causing approximately 80% of all cases of osteomyelitis with a broad host range in coagulase-positive staphylococci and is one of the leading causes of infections in oral and maxillofacial regions1, 2. Moreover, the rapidly increasing frequency of antibiotic-resistant bacteria has caused considerable alarm within the dental and medical community3, 4. Infections associated with this organism are extremely common and often life threatening; therefore, there is serious potential for S. aureus to cause increased morbidity and mortality in oral and maxillofacial regions3-6. To cause the infection, the bacteria do the followings: adhere and enter the host, multiply on or in the host tissues, resist host defences, and damage the host. All four processes must be accomplished in order to produce an infectious disease, and each is complex, involving several determinants. Bacterial attachment and penetration into underlying tissues play an important role in the initiation of infection. Some bacteria may penetrate tissue surfaces by producing lytic substances that break down the tissue surface, easing penetration to the deeper tissues, and other bacteria can penetrate through epithelial cells. It is likely that a variety of mechanisms are used by different organisms to penetrate the various tissue surfaces that they use as their portal of entry7. However lately, many authors have demonstrated the capability of the bacteria to attach on and invade the internal space of non-phagocytic eukaryotes8-13. On the other hand, it has been suggested that the continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts14-16. Several unrelated intracellular bacterial pathogens, including Listeria monocytogenes (L. monocytogenes), Shigella flexneri, Rickettsiae and Vaccinia virus, are known to share the ability to use actin polymerization as a driving force for intracellular movement, cell-to-cell spread and dissemination within infected tissues. While S. aureus has not traditionally been considered to be an intracellular pathogen, previous studies have revealed that S. aureus may be actively internalized by phagocytosis and are capable of intracellular survival in epithelial and endothelial cells17, 18. It has also been suggested that S. aureus interact with or binds to the cell surface of bovine

Effects of vasopressin on hyaluronate hydrolase activities and water permeability in the frog urinary bladder.

The involvement of enzymes catabolizing hyaluronic acid (hyaluronidase, beta-glucuronidase, N-acetyl-beta-D-hexosaminidase) in the hydroosmotic effect of vasopressin in the frog (Rana ridibunda) urinary bladder was studied. It was observed that vasopressin (50 nM), an agonist of V2 receptors, L-desamino-8-D-arginine-vasopressin (dDAVP, 1.5 microM) and forskolin (30 microM) activated the enzymes and caused their release into Ringer solution at the mucosal side, together with an increase in osmotic water flow. The effect of AVP on enzyme activity developed 10 min after the hydroosmotic response. Cytochalasin B (a specific inhibitor of actin filament elongation, 50 nM) blocked the hydroosmotic response to AVP; hyaluronate hydrolase activity increased in the bladder tissue but not in Ringer solution. It is suggested that the involvement of hyaluronate hydrolases in AVP's effect is mediated by a cAMP-dependent mechanism and provides favorable conditions for an increase in the permeability of glycosaminoglycan structures adjacent to the apical cell surface.

Initiation of apoptosis by actin cytoskeletal derangement in human airway epithelial cells.

Changes in epithelial cell shape can lead to cell death and detachment. Actin filaments are cleaved during apoptosis, but whether disruption in the actin cytoskeletal network, as one manifestation of cell shape change, can itself induce apoptosis is not known. We tested this hypothesis in the airway epithelial cell line 1HAEo(-) and in primary airway epithelial cells by preventing actin filament elongation with cytochalasin D or by aggregating actin filaments with jasplakinolide. Disruption of actin filament integrity promptly induced apoptosis in adherent epithelial cells within 5 h. Jasplakinolide-induced apoptosis did not disrupt focal adhesions, whereas cytochalasin D-induced apoptosis decreased focal adhesion protein expression and occurred despite ligation of the fibronectin receptor. Death induction was abrogated by the caspase inhibitors z-VAD-fmk and Ac-DEVD-cho but not by blocking the Fas (CD95) receptor. Whereas cytochalasin D--induced apoptosis was associated with cleavage of pro-caspase-8, jasplakinolide-induced apoptosis was not. Both agents induced formation of a death-inducing signaling complex. These data demonstrate that disruption of actin filament integrity with either cytochalasin D or jasplakinolide induces apoptosis in airway epithelial cells but by different mechanisms, and suggest that actin may be an early modulator of apoptotic commitment.

Biochemical and morphogenic effects of the interaction between protein kinase C-epsilon and actin in vitro and in cultured NIH3T3 cells.

Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.

Actin-Latrunculin A Structure and Function: DIFFERENTIAL MODULATION OF ACTIN-BINDING PROTEIN FUNCTION BY LATRUNCULIN A

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

Phorbol-ester-induced formation of actin filaments and microtubules: role of protein kinase C

Cell division and motility are generated by the cytoskeletal structures contained in networks of actin filaments and microtubules. Among the Ca2+-activated protein kinases, protein kinase C (PKC) can be activated with tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) (Kikkawaet al., 1983), which induces alterations in the cytoskeleton of cultured cells. To study the regulation of the cytoskeleton by PKC, we investigated the formation of actin filaments, microtubules, changes in cell shape and Ca2+signalling after treatment with TPA in unfertilised eggs of the sea urchinHemicentrotus pulcherrimus.Formation of actin filaments and microtubles was induced in the eggs by the TPA treatment. When the eggs were treated with an inactive TPA, 4α-PMA, these cytoskeletal changes did not occur. Moreover, elongation of actin filaments was inhibited by the Ca2+chelating agent BAPTA, but microtubule organisation was unaffected by intracellular Ca2+chelation in the TPA-treated egg. TPA is known to induce alkalisation in sea urchin eggs (Swann & Whitaker, 1985) and when the eggs are treated with TPA in Na+-free seawater, the rise in intracellular pH (pHi) is depressed. However, elongation of actin filaments and microtubules occurred under these conditions. An increase in pHiis considered to be essential for triggering the bundle of actin filaments (Begget al., 1982) and microtubule assembly (Schattenet al., 1992). These results show that actin and microtubule assemblies require activation of PKC, and that the formation of actin and microtubules may be related to the type of PKC isoform: Ca2+-dependent or -independent. Moreover, this mechanism is essentially independent of cytoplasmic alkalisation.

Corrigendum to: ‘Nucleation and elongation of actin filaments in the presence of high speed supernate from neutrophil lysates: modulating effects of Ca 2+ and phosphatidylinositol-4,5-bisphosphate’ : [Biochim. Biophys. Acta 1405 (1998) 85–98

Corrigendum to: ‘Nucleation and elongation of actin filaments in the presence of high speed supernate from neutrophil lysates: modulating effects of Ca 2+ and phosphatidylinositol-4,5-bisphosphate’ : [Biochim. Biophys. Acta 1405 (1998) 85–98

Cytoplasmic Ca2+ oscillation coordinates the formation of actin filaments in the sea urchin eggs activated with phorbol ester.

Changes in the intracellular Ca2+ concentration ([Ca2+]i) and the formation of actin filaments were investigated in unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus after activation with a phorbol ester, 12-O-tetradecanoyl phorbol13-acetate (TPA). Intracellular Ca2+ oscillation was observed using a fluorescent Ca2+ indicator dye, calcium green dextran. From about 20 to 80 min after the addition of TPA to 100 microM, there was a rise in [Ca2+]i, which was followed by Ca2+ oscillation. A change in [Ca2+]i in response to TPA was not observed in eggs that had been injected with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist. Therefore, long-term exposure to a high concentration of TPA seems to induce Ca2+ release via the IP3 pathway, as well as causing the release of diacylglycerol from membrane lipids. Moreover, the elongation of actin filaments occurred in the cytoplasm during the rise in [Ca2+]i. Actin filaments also formed when TPA-induced cytoplasmic alkalization was inhibited by exposure to Na(+)-free sea water. These results suggest that the observed cytoplasmic formation of actin filaments may be related to change in the cytoplasmic [Ca2(+)]i, and not intracellular pH, induced by TPA. These phenomena may be similar to the changes in actin construction that occur during cell cycle events.

Actin‐Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co‐align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co‐align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin‐binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly‐merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross‐linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.