Antiparallel Actin Filaments Research Articles

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.

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.

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.

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.

GTPase rho is involved in myosin-II-mediated contraction of pseudo-contractile rings and transport of vesicles in extracts of clam oocytes.

fusion while chelators with either a lower or higher binding constant do not (6). Therefore, the lack of a strong inhibitory effect of BAPTA on MLCK activity suggests that the binding constant for calmodulin lies outside the range of calcium fluctuations reported for BAPTA (1). The generation of sliding forces between anti-parallel actin filaments is a well-established activity of bipolar filaments of myosin-II. Therefore, the observation that actin filaments selforganized and moved in an anti-parallel fashion in these extracts fits current models of the contractile ring. These data support models in which the bundling of actin filaments involves a calcium-sensitive step. Calcium signaling through Rho G-protein pathways, which modulate the actin cytoskeleton, is the most likely mechanism by which cytosolic Ca controls actin bundle formation (7). The involvement of actin bundling activity in the formation of contractile rings has yet to be established, although these data raise such a possibility. Supported by NSF grant IBN-0131470 and MBL Shifman award to RFT.

The three-dimensional structure of a vertebrate wide (slow muscle) Z-band: lessons on Z-band assembly

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (∼30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (∼100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably α-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.

Three-Dimensional Structure of a Vertebrate Muscle Z-band: Implications for Titin and α-Actinin Binding

The Z-band in vertebrate striated muscles, mainly comprising actin filaments, α-actinin, and titin, serves to organise the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Different Z-band thicknesses, formed from different numbers of zigzag crosslinking layers and found in different fibre types, are thought to be associated with the number of repetitive N-terminal sequence domains of titin. In order to understand myofibril formation it is necessary to correlate the ultrastructures and sequences of the actin filaments, titin, and α-actinin in characteristic Z-bands. Here electron micrographs of the intermediate width, basketweave Z-band of plaice fin muscle have been subject to a novel 3D reconstruction process. The reconstruction shows that antiparallel actin filaments overlap in the Z-band by about 22–25 nm. There are three levels of Z-links (probably α-actinin) in which at each level two nearly diametrically opposed links join an actin filament to two of its antiparallel neighbours. One set of links is centrally located in the Z-band and there are flanking levels orthogonal to this. A 3D model of the observed structure shows how Z-bands of different widths may be formed and it provides insights into the structural arrangements of titin and α-actinin in the Z-band. The model shows that the two observed symmetries in different Z-bands, c2 and p121, may be attributed respectively to whether the number of Z-link levels is odd or even.

Heavy Meromyosin Induces Sliding Movements between Antiparallel Actin Filaments

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear.

The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.