Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane-associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.     

WH2 and proline‐rich domains of WASP‐family proteins collaborate to accelerate actin filament elongation

WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.     

Dynamin2 and cortactin regulate actin assembly and filament organization

The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Tropomodulin contains two actin filament pointed end-capping domains

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.     

Control of actin assembly and disassembly at filament ends

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.     

Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains

Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. Here, we show that N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, we reconstituted the propulsive motility of lipid-coated glass beads, using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation.     

Nucleation of polar actin filament assembly by a positively charged surface

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.     

Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin

Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.     

SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth

Organization of actin filaments into a well-organized sarcomere structure is critical for muscle development and function. However, it is not completely understood how sarcomeric actin/thin filaments attain their stereotyped lengths. In an RNAi screen in Drosophila primary muscle cells, we identified a gene, sarcomere length short (sals), which encodes an actin-binding, WH2 domain-containing protein, required for proper sarcomere size. When sals is knocked down by RNAi, primary muscles display thin myofibrils with shortened sarcomeres and increased sarcomere number. Both loss- and gain-of-function analyses indicate that SALS may influence sarcomere lengths by promoting thin-filament lengthening from pointed ends. Furthermore, the complex localization of SALS and other sarcomeric proteins in myofibrils reveals that the full length of thin filaments is achieved in a two-step process, and that SALS is required for the second elongation phase, most likely because it antagonizes the pointed-end capping protein Tropomodulin.     

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1.Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin.Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin.Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4.Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4.Actin and ushers actin as a Profilin.Actin complex onto growing barbed filament ends.     

The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.     

Characterization of engineered actin binding proteins that control filament assembly and structure

Background

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.

Methodology/Principal Findings

We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel “reduced genetic code” phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.

Conclusions/Significance

From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.     

The function of plakophilin 1 in desmosome assembly and actin filament organization

Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes. To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo. When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane. This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system. Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments. This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain. Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton.     

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.     

Actin-dependent cell elongation in teleost retinal rods: requirement for actin filament assembly

Teleost retinal rods elongate when exposed to light. Elongation is mediated by a narrow necklike region called the myoid. In the cichlid Sarotherodon mossambicus, the rod inner segment (composed of the myoid with adjacent ellipsoid) increases in length from 12 micrometers in the dark to 41 micrometers in the light. Long light-adapted myoids contain longitudinally oriented microtubules and bundles of parallel 60-A filaments that we have identified as actin by their ability to bind myosin subfragment 1. In short dark-adapted myoids, only microtubules are recognizable. Colchicine experiments reveal that light-induced rod elongation can occur in the absence of myoid microtubules. Intraocular injections of colchicine at concentrations that disrupt virtually all rod myoid microtubules do not block rod elongation. However, rod elongation is blocked by intraocular injections of cytochalasin B or cytochalasin D. The hierarchy of effectiveness of these drugs is consistent with their effectiveness in inhibiting actin assembly and in disrupting other actin-dependent motile processes. On the basis of ultrastructural observations and the results of these inhibitor studies, we propose that the forces responsible for rod elongation are dependent not on microtubules but on actin filament assembly.     

WH2 domain: a small, versatile adapter for actin monomers

The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins that usually contain one of the few known actin-binding motifs. WH2 domain (WASP homology domain-2) is a approximately 35 residue actin monomer-binding motif, that is found in many different regulators of the actin cytoskeleton, including the beta-thymosins, ciboulot, WASP (Wiskott Aldrich syndrome protein), verprolin/WIP (WASP-interacting protein), Srv2/CAP (adenylyl cyclase-associated protein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in beta-thymosin's interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain-containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer-binding motif that existed before the divergence of fungal and animal lineages.