Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Myosin-1C differentially displaces tropomyosin isoforms altering their inhibition of motility.

Force generation and motility by actomyosin in non-muscle cells are spatially regulated by ∼40 tropomyosin (Tpm) isoforms. The means by which Tpms are targeted to specific cellular regions and the mechanisms that result in differential activity of myosin paralogs are unknown. We show that Tpm3.1 and Tpm1.7 inhibit Myosin-IC (Myo1C), with Tpm1.7 more effectively reducing the number of gliding filaments compared to Tpm3.1. Strikingly, cosedimentation and fluorescence microscopy assays revealed that Tpm3.1 is displaced from actin by Myo1C and not by myosin-II. In contrast, Tpm1.7 is only weakly displaced by Myo1C. Unlike other characterized myosins, Myo1C motility is inhibited by Tpm when the Tpm-actin filament is activated by myosin-II. These results point to a mechanism for exclusion of myosin-I paralogs from cellular Tpm-decorated actin filaments that are activated by other myosins. Additionally, our results suggest a potential mechanism for myosin-induced Tpm sorting in cells.

A solution to the long-standing problem of actin expression and purification.

Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Crystal Structure of Leiomodin 2 in Complex with Actin: A Structural and Functional Reexamination.

Leiomodins (Lmods) are a family of actin filament nucleators related to tropomodulins (Tmods), which are pointed end-capping proteins. Whereas Tmods have alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, ABS2), Lmods lack TMBS2 and half of ABS1, and present a C-terminal extension containing a proline-rich domain and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain that is absent in Tmods. Most of the nucleation activity of Lmods resides within a fragment encompassing ABS2 and the C-terminal extension. This fragment recruits actin monomers into a polymerization nucleus. Here, we revise a recently reported structure of this region of Lmod2 in complex with actin and provide biochemical validation for the newly revised structure. We find that instead of two actin subunits connected by a single Lmod2 polypeptide, as reported in the original structure, the P1 unit cell contains two nearly identical copies of actin monomers, each bound to Lmod2's ABS2 and WH2 domain, with no electron density connecting these two domains. Moreover, we show that the two actin molecules in the unit cell are related to each other by a local twofold noncrystallographic symmetry axis, a conformation clearly distinct from that of actin subunits in the helical filament. We further find that a proposed actin-binding site within the missing connecting region of Lmod2, termed helix h1, does not bind actin in vitro and that the electron density assigned to it in the original structure corresponds instead to a WH2 domain with opposite backbone directionality. Polymerization assays using Lmod2 mutants of helix h1 and the WH2 domain support this conclusion. Finally, we find that deleting the C-terminal extension of Lmod1 and Lmod2 results in an approximately threefold decrease in the nucleation activity, which is only partially accounted for by the lack of the WH2 domain.

Hook Adaptors Induce Unidirectional Processive Motility by Enhancing the Dynein-Dynactin Interaction.

Cytoplasmic dynein drives the majority of minus end-directed vesicular and organelle motility in the cell. However, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Recent work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. Of particular interest, studies in fungal systems have implicated Hook proteins in the regulation of microtubule motors. Here we investigate the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. We used optogenetic approaches to specifically recruit Hook proteins to organelles and observed rapid transport of peroxisomes to the perinuclear region of the cell. This rapid and efficient translocation of peroxisomes to microtubule minus ends indicates that mammalian Hook proteins activate dynein rather than kinesin motors. Biochemical studies indicate that Hook proteins interact with both dynein and dynactin, stabilizing the formation of a supramolecular complex. Complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of end-binding (EB) proteins but cannot bind directly to microtubules. Single-molecule motility assays using total internal reflection fluorescence microscopy indicate that both Hook1 and Hook3 effectively activate cytoplasmic dynein, inducing longer run lengths and higher velocities than the previously characterized dynein activator bicaudal D2 (BICD2). Together, these results suggest that dynein adaptors can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking.

Glia maturation factor (GMF) interacts with Arp2/3 complex in a nucleotide state-dependent manner.

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 µM affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.

Myosin-I Synergizes with Arp2/3 Complex to Enhance Pushing Forces of Branched Actin Networks.

Myosin-Is colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by Arp2/3 complex on the surface of beads coated with myosin-I and the WCA domain of N-WASP. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Remarkably, myosin-I triggered symmetry breaking and comet-tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy.

Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins.

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.

Molecular mechanism of Arp2/3 complex inhibition by Arpin.

Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin's mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains.

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.

Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein.

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA.

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.

Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism.

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo-electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

Mechanism of actin N-terminal acetylation.

About 80% of human proteins are amino-terminally acetylated (Nt-acetylated) by one of seven Nt-acetyltransferases (NATs). Actin, the most abundant protein in the cytoplasm, has its own dedicated NAT, NAA80, which acts posttranslationally and affects cytoskeleton assembly and cell motility. Here, we show that NAA80 does not associate with filamentous actin in cells, and its natural substrate is the monomeric actin-profilin complex, consistent with Nt-acetylation preceding polymerization. NAA80 Nt-acetylates actin-profilin much more efficiently than actin alone, suggesting that profilin acts as a chaperone for actin Nt-acetylation. We determined crystal structures of the NAA80-actin-profilin ternary complex, representing different actin isoforms and different states of the catalytic reaction and revealing the first structure of NAT-substrate complex at atomic resolution. The structural, biochemical, and cellular analysis of mutants shows how NAA80 has evolved to specifically recognize actin among all cellular proteins while targeting all six actin isoforms, which differ the most at the amino terminus.

Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates.

CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3' end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFNalpha, IL-6, IFNgamma and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFNgamma-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.

A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity.

Cytoplasmic dynein is the major minus-end-directed microtubule-based motor in cells. Dynein processivity and cargo selectivity depend on cargo-specific effectors that, while generally unrelated, share the ability to interact with dynein and dynactin to form processive dynein-dynactin-effector complexes. How this is achieved is poorly understood. Here, we identify a conserved region of the dynein Light Intermediate Chain 1 (LIC1) that mediates interactions with unrelated dynein-dynactin effectors. Quantitative binding studies map these interactions to a conserved helix within LIC1 and to N-terminal fragments of Hook1, Hook3, BICD2, and Spindly. A structure of the LIC1 helix bound to the N-terminal Hook domain reveals a conformational change that creates a hydrophobic cleft for binding of the LIC1 helix. The LIC1 helix competitively inhibits processive dynein-dynactin-effector motility in vitro, whereas structure-inspired mutations in this helix impair lysosomal positioning in cells. The results reveal a conserved mechanism of effector interaction with dynein-dynactin necessary for processive motility.

How Leiomodin and Tropomodulin use a common fold for different actin assembly functions.

How proteins sharing a common fold have evolved different functions is a fundamental question in biology. Tropomodulins (Tmods) are prototypical actin filament pointed-end-capping proteins, whereas their homologues, Leiomodins (Lmods), are powerful filament nucleators. We show that Tmods and Lmods do not compete biochemically, and display similar but distinct localization in sarcomeres. Changes along the polypeptide chains of Tmods and Lmods exquisitely adapt their functions for capping versus nucleation. Tmods have alternating tropomyosin (TM)- and actin-binding sites (TMBS1, ABS1, TMBS2 and ABS2). Lmods additionally contain a C-terminal extension featuring an actin-binding WH2 domain. Unexpectedly, the different activities of Tmods and Lmods do not arise from the Lmod-specific extension. Instead, nucleation by Lmods depends on two major adaptations-the loss of pointed-end-capping elements present in Tmods and the specialization of the highly conserved ABS2 for recruitment of two or more actin subunits. The WH2 domain plays only an auxiliary role in nucleation.