Formation Of Actin Filament Networks Research Articles

A “Primer”-Based Mechanism Underlies Branched Actin Filament Network Formation and Motility

Cells use actin assembly to generate forces for membrane protrusions during movement [1] or, in the case of pathogens, to propel themselves in the host cells, in crude extracts [2], or in mixtures of actin and other purified proteins [3]. Significant progress has been made in understanding the mechanism of actin-based motility at a macroscopic level by using biomimetic systems in vitro [4-6]. Here, we combined such a system with evanescent wave microscopy to visualize Arp2/3-mediated actin network formation at single-actin-filament resolution. We found that actin filaments that we call "primers" determine the origin of the autocatalytic and propagative formation of the actin network. In the presence of capping protein, multiple "primers" generate independent networks that merge around the object to form an outer "shell" made of entangled and capped filaments. Simultaneously, newly created filaments on the surface of the particle initiate mechanical stress, which develops until symmetry breaking. Our results and extensive modeling support that the stress, which releases into propulsive forces [7], is controlled not by any specific orientation of actin filaments toward the nucleation sites but only by new monomers added near the load surface.

HA2 from the Helicoverpa armigera nucleopolyhedrovirus: a WASP-related protein that activates Arp2/3-induced actin filament formation

Filamentous actin is required for the productive replication of lepidopteran nucleopolyhedroviruses. We have demonstrated that nucleocapsids of the Helicoverpa armigera nucleopolyhedrovirus (HearNPV) are capable of nucleating actin polymerization in vitro in a dose-dependent manner. Actin polymerization is the main mechanism used in cell locomotion and is also utilized by the Listeria bacteria and by vaccinia virus for intracellular and intercellular movements. The WASP family of proteins has been shown to stimulate the assembly of branched actin filaments by the Arp2/3 complex. The process is conserved in eukaryotic cells. HearNPV ORF 2 (HA2), a WASP homologue, could nucleate branched actin filaments in the presence of Arp2/3 complex in vitro. We also demonstrate that HA2 co-localizes with Arp2/3 complex in the nucleus of infected cells, suggesting that HA2 and Arp2/3 complex are involved in nuclear actin polymerization. In summary, HA2 activates Arp2/3-induced actin filament network formation in vitro and in vivo.

Sequential Interaction of Actin-related Proteins 2 and 3 (Arp2/3) Complex with Neural Wiscott-Aldrich Syndrome Protein (N-WASP) and Cortactin during Branched Actin Filament Network Formation

The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin. Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.

Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins.

Actin filament assembly and turnover drive many forms of cellular motility, particularly extension of the leading edge of locomoting cells and rocketing of pathogenic microorganisms through host cell cytoplasm. De novo nucleation of actin filaments appears to be required for these movements. A complex of seven proteins called Arp2/3 complex is the best characterized cellular initiator of actin filament nucleation. Arp2/3 complex is intrinsically inactive, relying on nucleation promoting factors for activation. WASp/Scar family proteins are prominent cellular nucleation promoting factors. They bring together an actin monomer and Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction. WASp and N-WASP are intrinsically autoinhibited, and their activity is regulated by Rho-family GTPases such as Cdc42, membrane polyphosphoinositides, WIP/verprolin, and SH3 domain proteins. These interactions provide a final common pathway for many signaling inputs to regulate actin polymerization. Microorganisms either activate Arp2/3 complex directly or usurp N-WASP to initiate actin polymerization.

Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation.

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.

Effects of Chlorpromazine on Actin Polymerization: Slackening of Filament Elongation and Filament Annealing

We have analyzed the effect of chlorpromazine (CPZ) on pure actin. We have found that CPZ quenches Trp-79 and Trp-86 fluorescence and, in agreement with an earlier report on conventional actin, inhibits actin polymerization, lowering the extent of polymerization. Moreover, novel polymerization data are presented indicating that CPZ decreases the maximum polymerization rate in a dose-dependent manner. The assembly inhibition results from the slackening of oligomer formation during the early stages of polymerisation, of filament elongation and of filament annealing. Finally, CPZ strongly inhibits actin filament network formation.

Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon.

Actin filament organization may play an important role in the maintenance of differentiated functions in epithelial cells. We previously reported our success in inducing and maintaining gap junctions, which are two kinds of differentiated function, in primary rat hepatocytes cultured with 2% DMSO and 10-7 M glucagon. In the present study, we demonstrated the formation of actin filament networks in the hepatocytes cultured with 2% DMSO and 10-7 M glucagon. Actin filaments in hepatocytes cultured in medium with only 2% DMSO added from 96 h after plating were concentrated under the plasma membrane and were observed to be circumferential. In hepatocytes cultured in the medium with both 2% DMSO and 10-7 M glucagon added from 96 h, not only the circumferential actin filaments but also the formation of actin filament networks were observed and the networks developed well with time in culture. The networks were observed as a dome-like structure under the cell face and terminated at the circumferential actin filaments. They were composed of electron-dense star-like vertices connected by microfilament bundles of varying length and were also very sensitive to the actin disruptor cytochalasin B. However, during the network formation, there were no significant increases in the amounts of actin protein and mRNA. The actin filament networks of the hepatocytes in this culture system might be closely related to the maintenance of differentiated functions.

Talin Can Crosslink Actin Filaments into Both Networks and Bundles

The talin–actin interaction was examined by using negative staining and cosedimentation assays. At pH 6.4 and low ionic strength, talin extensively crosslinked actin filaments into both networks and bundles. The bundles consist of parallel actin filaments with a center-to-center distance of 13 nm, and talin crossbridges spaced at 36-nm intervals along the bundles. As pH was increased stepwise from 6.4 to 7.3, talin's bundling activity was decreased first, then its networking activity. Qualitatively similar results were obtained at pH 6.4 by increasing ionic strength. Chemical crosslinking indicated talin was present as a dimer from pH 6.4 to 7.3, with or without added KC1. The results show that talin can interact directly with actin filaments by formation of actin filament networks and bundles, with the bundles more sensitive to dissolution by increase in pH or ionic strength.

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

Cross-linking of actin filament networks by self-association and actin-binding macromolecules.

We have used low shear falling ball viscometry to measure the effects of actin purity, solution conditions, and cross-linking macromolecules on the formation of actin filament networks. Removal of minor contaminants from conventional muscle actin (Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871) by gel filtration (MacLean-Fletcher, S. and Pollard, T.D. (1980c) Biochem. Biophys. Res. Commun. 96, 18-27), greatly promotes the interaction of the filaments. Purified actin filaments form a gel (apparent viscosity greater than 12,000 cp) at approximately 2 mg/ml whereas approximately 12 mg/ml of conventional actin filaments have a viscosity of less than 400 cp. The apparent viscosity of the filaments depends on pH and the concentration of monovalent and divalent cations. The viscosity of purified action filaments is more sensitive to such variables when compared to that of conventional actin filaments. Together these experiments suggest that actin filament self-associations contribute to the stabilization of actin gels. A number of basic macromolecules, including aldolase, histones, lysozyme, polylysine, and RNase A can bind to and crosslink conventional actin filaments to form a gel. Since it is unlikely that all of these molecules are bound to actin in vivo, experimental approaches in addition to viscometry and sedimentation must be applied to prove that a given protein functions as an actin cross-linking protein in the cell.

Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association

Gel filtration of depolymerized conventionally purified muscle actin separates from the actin monomers a fraction of minor contaminants with a Stokes' radius of 4.7 nm which has the ability to block actin filament network formation. On the basis of heat and trypsin sensitivity, this inhibitory activity appears to be a protein. The inhibitory activity binds to actin filaments and reduces their low shear viscosity by up to 99% in a concentration dependent fashion while reducing polymerization to only a minor extent.

Mechanism of action of cytochalasin B on actin

Substoichiometric cytochalasin B (CB) inhibits both the rate of actin polymerization and the interaction of actin filaments in solution. The polymerization rate is reduced by inhibition of actin monomer addition to the “barbed” end of the filaments where monomers normally add more rapidly. 2 μM CB reduces the polymerization rate by up to 90%, but has little effect on the rate of monomer addition at the slow (“pointed”) end of the filaments and no effect on the rate of filament annealing. Under most ionic conditions tested, 2 μM CB reduces the steady state high shear viscosity by 10–20% and increases the steady state monomer concentration by a factor of 2.5 or less. In addition to the effects on the polymerization process, 2 μM CB strongly reduces the low shear viscosity of actin filaments alone and actin filaments cross-linked by a variety of macromolecules. This may be due to inhibition of actin filament-filament interactions which normally contribute to network formation. Since the inhibition of monomer addition and of actin filament network formation have approximately the same CB concentration dependence, a common CB binding site, probably the barbed end of the filament, may be responsible for both effects.