Antiparallel Actin Research Articles

Tension formation of a reconstructed nonmuscle sarcomere.

The interactions between actin and nonmuscle myosin 2 enable tension dependent processes in cells such as migration and adhesion. Although cortical actomyosin networks have been described as mostly disordered, several recent super resolution light microscopy studies have shown evidence for sarcomeric arrangement of actin and myosin in various cell types. Despite their resemblance to muscle sarcomeres where bipolar myosin filaments contract antiparallel actin fibers to bring about shortening, these structures are not as easily amenable to mechanical studies due to their subcellular scale and localization. To address this gap, we reconstituted a synthetic sarcomere using laser guided micropatterning of the actin elongator formin to polymerize antiparallel actin bundles. By adding nonmuscle 2 paralogs, the tension formation and shortening of these arrangements was observed using TIRF microscopy and quantified by tracking fluorescently labeled myosin filaments. Additionally, a FRET based force-sensor was added to the myosin tail and formin, elucidating the tension formation in actin and myosin filaments. Both actin and myosin displayed features resembling isometric tension like force generation within minutes after the start of the experiment, while shortening and processive motility of myosin could be observed more than 1 hour later. This allows us to propose a mechanism of tension formation inside cells, where isometric tension of 2 pNs is mediated by sliding individual actin filaments within a network of bundles, pushing against their bundling and independently of their length. This force generation mechanism at the myosin filament level would enable cells to mediate tension and contractility over the entire cortex, while deformations occur much slower, such as during the formation of a cytokinetic ring.

Cryo-ET reveals sarcomere structures at molecular resolution.

Muscles underpin movement and heart function. Individual muscle fibers can be very large syncytia of up to several centimeters in length and are comprised of the force-generating and load-bearing devices of muscles called sarcomeres. Contraction and relaxation of sarcomeres relies on the sliding between two types of filaments—the thin filament (comprising mainly F-actin, tropomyosin, and troponin) and the thick myosin filament. In addition, several other proteins are involved in the contraction mechanism and their mutational malfunction can lead to debilitating and even life-threatening diseases. In my presentation, I will explain how we managed to obtain high-resolution structures of native sarcomeres using cryo-electron tomography (cryo-ET). Our cryo-ET reconstructions reveal molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band and Z-disc and demonstrate that α-actinin cross-links antiparallel actin filaments by forming doublets with 6 nm spacing. Structures of myosin, tropomyosin, actin, and nebulin at up to 4.5 Å further reveal two conformations of “double-headed” myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. The structures provide high-resolution details of the interaction between nebulin and actin, demonstrating the stabilising role of nebulin. Unexpectedly, nebulin does not interact with myosin or tropomyosin, but with a troponin-T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

The molecular basis for sarcomere organization in vertebrate skeletal muscle

Summary Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 A further reveal two conformations of the double-head myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

The molecular basis for sarcomere organization in vertebrate skeletal muscle

SummarySarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the “double-head” myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

Existence and uniqueness of solutions for a model of non-sarcomeric actomyosin bundles

The model for disordered actomyosin bundles recently derived in [6]includes the effects of cross-linking of parallel and anti-parallel actin filaments,their polymerization and depolymerization, and, most importantly, the interactionwith the motor protein myosin, which leads to sliding of anti-parallel filamentsrelative to each other. The model relies on the assumption that actin filaments areshort compared to the length of the bundle. It is a two-phase model which treatsactin filaments of both orientations separately. It consists of quasi-stationary forcebalances determining the local velocities of the filament families and of transportequation for the filaments. Two types of initial-boundary value problems areconsidered, where either the bundle length or the total force on the bundle areprescribed. In the latter case, the bundle length is determined as a free boundary.Local in time existence and uniqueness results are proven. For the problem with givenbundle length, a global solution exists for short enough bundles. For small prescribedforce, a formal approximation can be computed explicitly, and the bundle lengthtends to a limiting value.

Structure of muscle α-actinin: Insights into its regulation and Z-disk assembly

α-Actinin is the major component of the Z-disk, where it cross-links actin filaments from adjacent sarcomeres. It is an antiparallel dimer of 200 kDa, containing in each subunit an N-terminal actin binding domain (ABD), a central rod domain assembled from spectrin-like repeats that mediate the antiparallel assembly, and a C-terminal calmodulin-like (CaM-like) domain with 4 EF-hand motifs. Additionally to actin filaments, α-actinin binds multiple other cytoskeletal and signalling proteins. In striated muscle, the tightly defined numbers of α-actinin crosslinks between the antiparallel actin filaments at the Z-disk are organised by specific binding sites on the giant molecular blueprint of the sarcomere, titin. These titin Z-repeats contain a short, hydrophobic, α-actinin binding motif. To achieve ordered cytoskeletal assemblies, the binding properties of α-actinin must be tightly spatiotemporally regulated, in muscle α-actinin its actin and titin binding properties are regulated by phosphoinositide. Biochemical analyses led to propose previously that the α-actinin - titin interaction is regulated by an intramolecular mechanism, where the short sequence between the ABD and the rod interacts with the CaM-like domain in a pseudoligand complex, acting effectively as an intramolecular autoinhibitor. Here, we present the first complete crystallographic structure of sarcomeric human α-actinin complemented by small angle X-ray scattering data, electron-electron paramagnetic resonance, biochemical and in vivo cell biophysics studies of structure-informed mutants, which give insight into its molecular assembly and Z-disk architecture as well as into the mechanism of α-actinin function and regulation.

Directed Actin Assembly and Contractility

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture is maintained in a dynamic steady-state through regulated assembly and disassembly. We have developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays (Reymann, Nat Mat, 2010). These actin templates were used to evaluate the response of oriented actin structures to myosin-induced contractility. We determine that myosins selectively contract and disassemble anti-parallel actin structures while parallel actin bundles remain unaffected. In addition, the local distribution of nucleation sites and the resulting orientation of actin filaments regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly reveals how the dynamics of the cellular actin cytoskeleton is spatially controlled by actomyosin contractility. Further application of the micropatterning method will be presented in particular recent data on the reconstitution of a lamellipodium-type of actin organization and the fabrication of three-dimensional electrical connections by means of directed actin self-organization.

FHOD1 regulates stress fiber organization by controlling transversal arc and dorsal fiber dynamics

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior.

Disulfide Cross-Linked Antiparallel Actin Dimer

Oxidation of actin monomer (G-actin) with copper o-phenanthroline resulted in a rapid, high yield of disulfide cross-linked dimer. The cross-link is due to an intermolecular disulfide bond between actin Cys374 of each molecule, resulting in a tail-to-tail, i.e., antiparallel, actin dimer. Analytical ultracentrifugation profiles of G-actin can be ascribed to the existence of actin monomers with very little, if any, dimer. Thus, actin dimers are not energetically favorable, indicating that cross-linked dimers are formed during random diffusional collisions. On the other hand, a similar oxidation of actin polymer (F-actin) resulted in a much lower yield of the cross-linked actin dimer that showed no sign of leveling off. Therefore, it is proposed that the cross-linked dimer from actin polymer is due to collisional complexes of actin monomers that are in equilibrium with the polymer during actin treadmilling. These results account for the reported observation that during the early stages of actin polymerization (where the actin monomer concentration is high) cross-linked antiparallel actin dimers are formed in relatively high yield whereas none are formed at later stages of polymerization. These findings raise questions concerning the validity of the antiparallel actin dimer model of in vitro actin polymerization that is based on the assumption that the ability to form cross-linked actin dimers implies the existence of stable dimers.

Nonmuscle myosin II exerts tension but does not translocate actin in vertebrate cytokinesis

During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.

An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells

The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells.

The Effect of Toxofilin on the Structure of Monomeric Actin

University of Pécs, Faculty of Medicine, Department of Biophysics, 7624 Pécs, Szigeti street 12, Hungary The actin cytoskeleton of eukaryotic cells plays a key role in many processes, including motility and cytokinesis. The structure and dynamics of the cytoskeleton are regulated by a large number of proteins that interact with monomeric and/or filamentous actins. Toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. One of the expressed proteins of T. gondii is the 27 kDa-sized toxofilin. The 245 amino acid long protein is a monomeric actin-binding protein involved in the host invasion. It can bind to actin monomers and to the ends of the actin filaments as well. The protein has three actin-binding sites which makes it capable to interact with an antiparallel actin dimer. In our work we studied the effect of the actin-binding site of toxofilin69-196 on the monomeric actin. We determined the affinity of toxofilin to the actin monomer with fluorescence anisotropy measurement (KD = 1.3 µM). The flourescence of the actin bound ε-ATP was quenched with acrylamide in the presence or absence of toxofilin. In the presence of toxofilin the accessibility of the bound ε-ATP decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. The results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites.

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30–50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.

Structural Memory in the Contractile Ring Makes the Duration of Cytokinesis Independent of Cell Size

Cytokinesis is accomplished by constriction of a cortical contractile ring. We show that during the early embryonic divisions in C. elegans, ring constriction occurs in two phases--an initial phase at a constant rate followed by a second phase during which the constriction rate decreases in proportion to ring perimeter. Cytokinesis completes in the same amount of time, despite the reduction in cell size during successive divisions, due to a strict proportionality between initial ring size and the constant constriction rate. During closure, the myosin motor in the ring decreases in proportion to perimeter without turning over. We propose a "contractile unit" model to explain how the ring retains a structural memory of its initial size as it disassembles. The scalability of constriction may facilitate coordination of mitotic events and cytokinesis when cell size, and hence the distance traversed by the ring, varies during embryogenesis and in other contexts.

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69-196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.

Crystal Structure of Polymerization-Competent Actin

All actin crystal structures reported to date represent actin complexed or chemically modified with molecules that prevent its polymerization. Actin cleaved with ECP32 protease at a single site between Gly42 and Val43 is virtually non-polymerizable in the Ca-ATP bound form but remains polymerization-competent in the Mg-bound form. Here, a crystal structure of the true uncomplexed ECP32-cleaved actin (ECP-actin) solved to 1.9 Å resolution is reported. In contrast to the much more open conformation of the ECP-actin's nucleotide binding cleft in solution, the crystal structure of uncomplexed ECP-actin contains actin in a typical closed conformation similar to the complexed actin structures. This unambiguously demonstrates that the overall structure of monomeric actin is not significantly affected by a multitude of actin-binding proteins and toxins. The invariance of actin crystal structures suggests that the salt and precipitants necessary for crystallization stabilize actin in only one of its possible conformations. The asymmetric unit cell contains a new type of antiparallel actin dimer that may correspond to the “lower dimer” implicated in F-actin nucleation and branching. In addition, symmetry-related actin-actin contacts form a head to tail dimer that is strikingly similar to the longitudinal dimer predicted by the Holmes F-actin model, including a rotation of the monomers relative to each other not observed previously in actin crystal structures.

Cytokinesis Goes Polo

Temporal and spatial coordination of cytokinesis with chromosome segregation is key for successful cell division, but it is poorly understood. A recent article in Science by Pellman and coworkers (Yoshida et. al., 2006) reveals how the yeast polo-like kinase Cdc5 triggers Rho1/RhoA activation and the assembly of the contractile actin ring during anaphase.

A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.