Structural Basis of Actin Filament Nucleation by Tandem W Domains

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Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.     

Latrunculin A Accelerates Actin Filament Depolymerization in Addition to Sequestering Actin Monomers

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Aip1 Promotes Actin Filament Severing by Cofilin and Regulates Constriction of the Cytokinetic Contractile Ring

Aip1 (actin interacting protein 1) is ubiquitous in eukaryotic organisms, where it cooperates with cofilin to disassemble actin filaments, but neither its mechanism of action nor its biological functions have been clear. We purified both fission yeast and human Aip1 and investigated their biochemical activities with or without cofilin. Both types of Aip1 bind actin filaments with micromolar affinities and weakly nucleate actin polymerization. Aip1 increases up to 12-fold the rate that high concentrations of yeast or human cofilin sever actin filaments, most likely by competing with cofilin for binding to the side of actin filaments, reducing the occupancy of the filaments by cofilin to a range favorable for severing. Aip1 does not cap the barbed ends of filaments severed by cofilin. Fission yeast lacking Aip1 are viable and assemble cytokinetic contractile rings normally, but rings in these Δaip1 cells accumulate 30% less myosin II. Further, these mutant cells initiate the ingression of cleavage furrows earlier than normal, shortening the stage of cytokinetic ring maturation by 50%. The Δaip1 mutation has negative genetic interactions with deletion mutations of both capping protein subunits and cofilin mutations with severing defects, but no genetic interaction with deletion of coronin.     

Actin Filament Length Tunes Elasticity of Flexibly Cross-Linked Actin Networks

Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.     

A Highlights from MBoC Selection: Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and γ-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010 , MBoC 21: 2792–2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.     

Abl2/Abl-related Gene Stabilizes Actin Filaments,Stimulates Actin Branching by Actin-related Protein 2/3 Complex,and Promotes Actin Filament Severing by Cofilin

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.     

Structure and Dynamics of the Actin Filament

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of γ-phosphate from ADP-P_i actin.     

A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

A Myopathy-linked Desmin Mutation Perturbs Striated Muscle Actin Filament Architecture

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.