Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.     

IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling

Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42 (refs 7, 8), which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. Here we demonstrate that IRSp53, a substrate for insulin receptor with unknown function, is the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, we found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex.     

Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.

Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the events occurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, uses actin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknown phosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck and the recruitment of the Ena/VASP family member N-WASP to the site of actin assembly. We also show that Nck and N-WASP are essential for the actin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actin polymerization at the plasma membrane.     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.     

Drosophila Spire is an actin nucleation factor

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.     

The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.     

Actin dynamics in the contractile ring during cytokinesis in fission yeast

Cytokinesis in many eukaryotes requires a contractile ring of actin and myosin that cleaves the cell in two. Little is known about how actin filaments and other components assemble into this ring structure and generate force. Here we show that the contractile ring in the fission yeast Schizosaccharomyces pombe is an active site of actin assembly. This actin polymerization activity requires Arp3, the formin Cdc12, profilin and WASP, but not myosin II or IQGAP proteins. Both newly polymerized actin filaments and pre-existing actin cables can contribute to the initial assembly of the ring. Once formed, the ring remains a dynamic structure in which actin and other ring components continuously assemble and disassemble from the ring every minute. The rate of actin polymerization can influence the rate of cleavage. Thus, actin polymerization driven by the Arp2/3 complex and formins is a central process in cytokinesis. Our studies show that cytokinesis is a more dynamic process than previously thought and provide a perspective on the mechanism of cell division.     

The dynamics of actin-based motility depend on surface parameters

In cells, actin polymerization at the plasma membrane is induced by the recruitment of proteins such as the Arp2/3 complex, and the zyxin/VASP complex. The physical mechanism of force generation by actin polymerization has been described theoretically using various approaches, but lacks support from experimental data. By the use of reconstituted motility medium, we find that the Wiskott Aldrich syndrome protein (WASP) subdomain, known as VCA, is sufficient to induce actin polymerization and movement when grafted on microspheres. Changes in the surface density of VCA protein or in the microsphere diameter markedly affect the velocity regime, shifting from a continuous to a jerky movement resembling that of the mutated 'hopping' Listeria. These results highlight how simple physical parameters such as surface geometry and protein density directly affect spatially controlled actin polymerization, and play a fundamental role in actin-dependent movement.     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate

The cortical cytoplasm, including the cleavage furrow, is largely composed of a network of actin filaments that is rigid even as it is extensively deformed during cytokinesis. Here we address the question of how actin-filament networks such as those in the cortex can be simultaneously rigid (solid-like) and fluid-like. Conventional explanations are that actin filaments rearrange by some combination of depolymerization and repolymerization; fragmentation and annealing of filaments; and inactivation and reestablishment of crosslinks between filaments. We describe the mechanical properties of a model system consisting of actin filaments and Acanthamoeba alpha-actinin, one of several actin crosslinking proteins found in amoeba and other cells. The results suggest another molecular mechanism that may account for the paradoxical mechanical properties of the cortex. When deformed rapidly, these mixtures are 40 times more rigid than actin filaments without alpha-actinin, but when deformed slowly these mixtures were indistinguishable from filaments alone. These time-dependent mechanical properties can be explained by multiple, rapidly rearranging alpha-actinin crosslinks between the actin filaments, a mechanism proposed by Frey-Wyssling to account for the behaviour of cytoplasm long before the discovery of cytoplasmic actin or alpha-actinin. If other actin-filament crosslinking proteins behave like Acanthamoeba alpha-actinin, this mechanism may explain how the cortex recoils elastically from small rapid insults but deforms extensively when minute forces are applied over long periods of time.     

Steps and fluctuations of Listeria monocytogenes during actin-based motility

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization. Although the biochemical and genetic aspects of Listeria motility have been intensely studied, biophysical data are sparse. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin. We occasionally observed smaller steps (<3 nm), as well as periods of motion with no obvious pauses. Clearly, bacteria do not sense cytoplasmic viscoelasticity because they fluctuate 20 times less than adjacent lipid droplets. Instead, bacteria bind their own actin 'tails, and the anchoring proteins can 'step' along growing filaments within the actin tail. Because positional fluctuations are unusually small, the forces of association and propulsion must be very strong. Our data disprove the brownian ratchet models and limit alternative models, such as the 'elastic' brownian ratchet or the 'molecular' ratchet.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

The maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel-sol' transformations result from the rearrangement of cortical actin-rich networks. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, alpha-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels rheologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.     

Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate

The actin-binding protein gelsolin requires micromolar concentrations of calcium ions to sever actin filaments, to potentiate its binding to the end of the filament and to promote the polymerization of monomeric actin into filaments. Because transient increases in both intracellular [Ca2+] and actin polymerization accompany the cellular response to certain stimuli, it has been suggested that gelsolin regulates the reversible assembly of actin filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cytoplasmic [Ca2+] and that once actin-gelsolin complexes form in the presence of Ca2+ in vitro, removal of free Ca2+ causes dissociation of only one of two bound actin monomers from gelsolin and the resultant binary complexes cannot sever actin filaments. The finding that cellular gelsolin-actin complexes can be dissociated suggests that a Ca2+-independent regulation of gelsolin also occurs. Here we show that, like the dissociation of profilin-actin complexes, phosphatidylinositol 4,5-bisphosphate, which undergoes rapid turnover during cell stimulation, strongly inhibits the actin filament-severing properties of gelsolin, inhibits less strongly the nucleating ability of this protein and restores the potential for filament-severing activity to gelsolin-actin complexes.     

Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.     

Phase transitions in the assembly of multivalent signalling proteins

Cells are organized on length scales ranging from ?ngstr?m to micrometres. However, the mechanisms by which ?ngstr?m-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.     

Structure and control of the actin regulatory WAVE complex

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-?ngstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.     

Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.     

Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.