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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 this helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.

A cryo-EM structure and functional study show that Arp2/3 complex activation requires NPF-mediated delivery of actin to both Arps., 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.

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.

A SAXS-based structural model is described for PICK1, a key player in AMPA receptor trafficking. It is shown that the acidic C-terminal tail of PICK1 is involved in autoinhibition and motility of PICK1-associated vesicle-like structures, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex., PICK1 is a modular scaffold implicated in synaptic receptor trafficking. It features a PDZ domain, a BAR domain, and an acidic C-terminal tail (ACT). Analysis by small- angle x-ray scattering suggests a structural model that places the receptor-binding site of the PDZ domain and membrane-binding surfaces of the BAR and PDZ domains adjacent to each other on the concave side of the banana-shaped PICK1 dimer. In the model, the ACT of one subunit of the dimer interacts with the PDZ and BAR domains of the other subunit, possibly accounting for autoinhibition. Consistently, full-length PICK1 shows diffuse cytoplasmic localization, but it clusters on vesicle-like structures that colocalize with the trans-Golgi network marker TGN38 upon deletion of either the ACT or PDZ domain. This localization is driven by the BAR domain. Live-cell imaging further reveals that PICK1-associated vesicles undergo fast, nondirectional motility in an F-actin–dependent manner, but deleting the ACT dramatically reduces vesicle speed. Thus the ACT links PICK1-associated vesicles to a motility factor, likely myosin, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex.

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.

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio VopL depends on its three W domains and dimerization through a unique VopL C-terminal domain (VCD). The VCD displays a novel all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment, but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

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.

Lmod is a muscle-specific actin nucleator that displays structural similarity to the filament pointed-end–capping protein, Tmod. The mechanisms of localizations of Lmod and Tmod in muscle sarcomeres are strikingly different. Lmod contributes to the organization of mature myofibrils through a mechanism that requires interaction with tropomyosin., Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.

Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actin-binding protein, leiomodin, that acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed end–capping protein tropomodulin: a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity threefold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, which suggests a role for leiomodin in the nucleation of tropomyosin-decorated filaments in muscles.

Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2′-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4–6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process.

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., Leiomodins and Tropomodulins are related, but have different functions; actin filament nucleation and pointed end capping, respectively. Here, the authors use structural, biochemical and cellular approaches to show how these different activities have evolved based on a common protein fold.

The antisense or antigene properties are exerted not only by natural oligonucleotides, but also by their different analogs. Among them, phosphorothioate oligonucleotides (PS-DNA), in which the sugar-phosphate backbone is modified due to substitution of the sulfur atom for one of the nonbridging oxygens, are much more resistant toward nucleases and, simultaneously, maintain good hybridization properties. However, the substitution induces the P-chirality of dinucleoside phosphorothioate moiety and even short PS-DNA synthesized using standard chemical methods exist as a mixture of hundreds or thousands of diastereoisomers. Diastereomerically pure oligomers of [PS]-d(CG)4 and [PS]-d(GC)4 series, obtained using the oxathiaphospholane method, were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. Obtained data allow us to postulate the formation of a strong intramolecular hydrogen bond with anionic sulfur atom as an acceptor. Homopurine [All-RP-PS]-oligos, but not [All-SP-PS]-oligos, form with the RNA templates extremely stable triplex structures, so far not described in the literature. Most likely, the triplexes are stabilized by hydrogen bonds or water bridges with the participation of sulfur atoms of internucleotide linkage. Notably, target RNA molecules are "arrested" by properly designed [All-RP-PS]-DNA probes at submicromolar concentration, and as the result, they are not recognized by reverse transcriptase.

Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.

Actin filament nucleation and branching by Arp2/3 complex is activated by nucleation-promoting factors (NPFs), whose C-terminal WCA region contains binding sites for actin (W) and Arp2/3 complex (CA). It is debated whether one or two NPFs are required for activation. Here, we present conclusive evidence in support of the two-NPF model and show that actin plays a crucial role in the interactions of two mammalian NPFs, N-WASP and WAVE2, with Arp2/3 complex. Competition between actin-WCA and glia maturation factor (GMF) for binding to Arp2/3 complex suggests that during activation the first actin monomer binds at the barbed end of Arp2. Based on distance constrains obtained by time-resolved fluorescence resonance energy transfer, we define the relative position of the two actin-WCAs on Arp2/3 complex and propose an atomic model of the 11-subunit transitional complex.

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.

PICK1 is a BAR domain protein, featuring additionally an N-terminal PDZ domain and an acidic C-terminal tail (ACT). PICK1 has been implicated in trafficking of several neuronal proteins, including the AMPA receptor. A recent article (Karlsen et al., 2015) reports a structural study of PICK1 using small angle X-ray scattering (SAXS). This work reaches different conclusions than a prior study from our laboratory that used a similar approach (Madasu et al., 2015). The new study, however, was apparently unaware of our work and did not address important contrary evidence.

New monomers, 5‘-O-DMT-deoxyribonucleoside 3‘-O-(2-thio-“spiro”-4,4-pentamethylene-1,3,2-oxathiaphospholane)s, were prepared and used for the stereocontrolled synthesis of PS−Oligos via the oxathia...

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

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.

Cell migration is a key step in tumor invasion and metastasis, involving dynamic reorganization of the actin cytoskeleton and membranes. BAR domain containing proteins have emerged as important regulators of the cytoskeleton that aid in coupling membrane and cytoskeleton dynamics to signaling pathways. IRSp53 is a BAR domain containing protein that is a potent inducer of lamellipodia- and filopodia-based cell motility under the control of Rho-family GTPases, making it a key player in tumor metastasis. However, little is known about the mechanisms of regulation of BAR domain-containing proteins in general and IRSp53 in particular. Here, we show a structural mechanism for IRSp53 regulation, which involves the release of autoinhibitory interactions by Cdc42. Central to this mechanism is a semi-conserved CRIB-PR motif that is rich in prolines (CRIB-PR) that joins the N-terminal BAR domain to the C-terminal SH3 domain. We have employed several biophysical techniques to confirm that both GTP-Cdc42 and the SH3 domain can bind to a CRIB-PR motif. Furthermore, we have used FRET to show IRSp53 undergoes a conformational change upon Cdc42 binding, consistent with the release of an autoinhibitory interaction. A crystal structure of a complex between activated Cdc42 and the CRIB-PR motif shows that proline-rich region binds to a region on Cdc42, not previously seen by conventional CRIB domains. Mutagenesis studies not only confirm this binding in solution and in cells, but also indicate that the SH3 domain is required for autoinhibition. In conclusion, we show a structural mechanism for IRSp53 regulation that involves a uniquely adapted CRIB motif that serves as a Cdc42-dependent switch for IRSp53 function, which helps to explain it's indispensible role in cell migration.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53) is a ubiquitous regulator of the actin cytoskeleton that mediates filopodia and lamellipodia formation under the control of Rho-family GTPases. IRSp53 is comprised of an N-terminal IRSp53/MIM homology domain (IMD) that senses negative membrane curvature and a central semi-CRIB motif followed by an SRC homology 3 (SH3) domain that binds to proline-rich regions of a wide range of actin regulators. Here, we present the crystal structure of a complex between activated CDC42 and the CRIB domain of IRSp53. Isothermal titration calorimetry (ITC) experiments confirm the binding of this domain to CDC42, and exclude the possibility of binding by inactive forms of CDC42 or activated forms of RAC1. Moreover, we demonstrate that the SH3 domain also binds to the CRIB domain, suggesting a mechanism of regulation where binding of CDC42 is required to release autoinhibition of the SH3 domain.

Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

The actin cytoskeleton is intimately involved in most cellular functions, including cell motility, endo/exocytosis and intracellular trafficking. These processes are characterized by rapid oscillations of actin polymerization/depolymerization under tight temporal and spatial regulation. Hundreds of G- and F-actin-binding proteins, along with signaling and scaffolding proteins regulate the assembly of actin networks. Among these proteins, filament nucleators play a critical role by determining the time and location for actin polymerization, as well as the specific structures of the actin networks that they generate. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin nuclei (small oligomers of 2-4 actin subunits), whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of Nucleation Promoting Factors (NPFs), Formins, Spire, Cobl, Lmod, VopL/VopF and TARP. Structural and functional studies are beginning to shed light on the diverse mechanisms used by these molecules to stabilize actin nuclei. Thus, with the exception of Formins known filament nucleators use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. The nucleation activities of these proteins vary significantly, and the most effective nucleators are not necessarily those with the largest number of W domains. We show that the inter-W linkers play a critical role in determining the nucleation activities of filament nucleators and the structures of the actin nuclei that they generate. Furthermore, we present evidence that a previously neglected factor, oligomerization, is a major determinant of filament nucleation activity and nuclei structure.

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 α-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with α-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.

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.

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.

Retroviral reverse transcriptases catalyze the synthesis of a double-stranded DNA copy of the RNA genome for integration into host chromosome. These enzymes possess three enzymatic activities essential for retrovims replication: an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase which synthesizes the second strand of the proviral DNA, and an RNase H which degrades RNA template after the synthesis of the first strand of the proviral DNA The importance of reverse transcriptase (RT) in the life cycle of retroviruses (e.g. HIV) makes the enzyme a preferred target for antiviral strategies [1].

The study of the actin filament is one of the major problems of structural biology. Uncontrollable polymerization has interfered with our ability to obtain crystals of the filament. Proteins that initiate actin polymerization in cells have the natural ability to stabilize multiple actin subunits into a filament-like conformation, allowing them to overcome the rate-limiting step in polymerization, i.e. the formation of actin dimers and trimers. 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. The W domain is a short (17–27 aa) actin-binding motif. A common architecture, found in filament nucleators such as Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. We have engineered a stable actin trimer stabilized by a tandem W domain hybrid construct that also includes filament barbed and pointed end capping elements. The structure of the actin trimer was first studied in solution using x-ray scattering. Different crystal forms of the trimer have been obtained. We will present these studies. In particular, we will show how tandem W domains stabilize a polymerization nucleus by organizing actin subunits into a filament-like conformation.Supported by NIH Grant HL086655

Summary 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.

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.

Leiomodin (Lmod) is related to tropomodulin (Tmod), and the two proteins have similar localization in striated muscle, i.e. near the M-line, in association with the pointed end of the actin filaments. However, in vitro the two proteins have fundamentally different activities: Lmod is a powerful actin filament nucleator, whereas Tmod caps the pointed end. Originally, it was assumed that the different domain organizations of the two proteins explained their different activities. Thus, Tmod has two tropomyosin (TM)- and two actin-binding sites, organized as: TMBS1-ABS1-TMBS2-ABS2. Lmod is longer, featuring a C-terminal extension that contains a Pro-rich region and a WH2 domain, which constitutes a third actin-binding site. The presence of the WH2 was considered to be the major feature distinguishing Lmod from Tmod. Here we show that this is not the case. Among the main findings are: 1) Lmod not only lacks TMBS2, but also ABS1, such that the entire region N-terminal to ABS2 has very little effect on nucleation, 2) The C-terminal extension of Lmod has also a limited effect on nucleation, and adding it to Tmod produces a very modest increase in nucleation, 3) Despite being relatively well conserved, the major feature distinguishing Lmod from Tmod is ABS2, consistent mostly of a Leu-rich repeat domain. Structural analysis shows that ABS2 can bind up to 3 actin subunits, and subtle differences between Lmod and Tmod dictate the affinities of their interactions with actin, and thus their roles in nucleation vs. capping. Understanding these differences allowed us to engineer an ABS2 Tmod-Lmod hybrid with nucleation activity equal to that of full-length Lmod.

The formation of new filaments is an essential, but kinetically unfavorable, step in actin cytoskeleton remodeling. In cells nucleation has to be catalyzed by proteins called nucleators that bring together at least two actin monomers and stabilize a polymerization nucleus. Among known filament nucleators Leiomodin (Lmod) is the only one specifically expressed in muscle cells. Lmod is related to the F-actin pointed-end capping protein tropomodulin (Tmod), with which it shares two actin-binding sites: a flexible N-terminal region and a leucine-rich repeat (LRR) domain. In addition, Lmod contains a unique C-terminal extension that features a WH2 domain, another actin-binding site.There are three muscle-type specific isoforms of Lmod: smooth muscle Lmod1, cardiac and striated muscle Lmod2, and the fetal isoform Lmod3. To date, only Lmod2 has been shown to nucleate actin filaments. Lmod1 is quite different in sequence from Lmod2 (37% identity, most of which is concentrated in the LRR). Here we demonstrate that despite these differences smooth muscle Lmod1 is also an actin filament nucleator. However its activity and mechanism of nucleation vary significantly from that of Lmod2.

NAA80 Nt-acetylates the six actin isoforms by specifically targeting the complex of actin-profilin among all cellular proteins., 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.

Arp2/3 complex is a ubiquitous actin filament nucleator comprised of seven proteins, two of which (Arp2 and Arp3) are related to actin. By itself Arp2/3 complex is inactive. It is activated by nucleation-promoting factors (NPFs) that contain a C-terminal WCA sequence. By bringing together actin and Arp2/3 complex NPFs catalyze a reaction that leads to the formation of a new actin filament branch bound at 70° angle to the side of a pre-existing filament. It is generally believed that the activation mechanism involves a conformational change within Arp2/3 complex that brings the two Arps close to one another, analogous to two parallel actin subunits of the actin filament. The two Arps, together with the actin monomer(s) delivered by NPFs, form a polymerization ‘seed’. However, the exact mechanism of activation remains unknown, and there is disagreement as to which subunits of Arp2/3 complex interacts with NPFs and how. Currently, there are two competing models of activation. The first model is based on small angle X-ray scattering of Arp2/3 complex bound to WCA region of the NPF and actin. According to this model only one NPF binds Arp2/3 complex and delivers the first actin monomer of the branch at the barbed end of the Arp2. The second model assumes that two NPF molecules are required for optimal activation, with the first actin monomer being delivered at the barbed end of Arp3. The studies in support of the latter mechanism do not directly analyzed binding of NPF carrying actin to Arp2/3 complex. Here, we present a study of the polymerization mechanism of the Arp2/3 complex that takes into consideration this critically missing factor, by using ITC and fluorescence to investigate the interaction of NPFs carrying actin with Arp2/3 complex.