Explore the vast range of titles available.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the “C-zone.” Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the ‘twitch’ response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s-1) matches the rate of Ca2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s-1) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s-1) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament—myosin head complex under physiological conditions.

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of El (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Downregulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.

It is widely accepted that contraction of skeletal muscle and the heart involves structural changes in actin-containing thin filaments to allow binding of myosin motors from neighbouring thick filaments, thus driving filament sliding; here, X-ray diffraction of single skeletal muscle cells reveals that this thin-filament mechanism can regulate muscle contraction against low load, but high-load contraction requires a second permissive step involving a structural change in the thick filament. A new take on muscle contraction It is thought that for the contraction of skeletal muscle and the heart, structural changes in the actin-containing thin filaments allow the binding of filaments to myosin motors from the neighbouring thick filaments that drives filament sliding. Yet this decades-old concept cannot account for the fact that, in resting muscle, myosin motors cannot bind to the thin filaments. Vincenzo Lombardi and colleagues test the hypothesis that a second permissive step for muscle shortening involves a structural change in the thick filament. Their analysis of single skeletal muscle cells reveals that the accepted thin-filament mechanism, involving a small fraction of constitutively 'ON' myosin motors, can regulate muscle contraction against low load. However, force generation against high load indeed requires change in the structure of the thick filaments, which under low load form an 'OFF' structure. Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding 1 , 2 , 3 . Here we show by synchrotron small-angle X-ray diffraction of frog ( Rana temporaria ) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched ‘OFF’) structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity 2 , 4 , 5 . This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched ‘ON’) myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding (1-3). Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity (2,4,5). This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

Muscle contraction relies on the coordinated activation of myosin motors from a folded-OFF state on the thick filament surface and their actin tracks on the thin filaments in response to calcium. Thick filaments contain distinct regulatory zones defined by the presence of myosin-binding protein C (MyBP-C) and titin super-repeats, but the control of myosin OFF/ON states within these zones has not been directly resolved. Here we do so, by fluorescence polarization microscopy (FPM). Using orientation-specific probes on myosin we show that folded-OFF motors are enriched in the MyBP-C–containing C zone in relaxed myofibrils. Under titin-based passive tension or partial calcium activation, active motors are enriched in the D zone at the filament tips, which lacks MyBP-C. Troponin probes further reveal that myosin enhances thin-filament activation in the region of filament overlap and drives activation into adjacent non-overlap regions. These findings uncover zone-specific control of myofilament activation within the sarcomere and establish FPM as a powerful tool for investigating disease-linked myofilament protein variants and therapeutic modulation.

Slow skeletal muscles maintain posture and produce graded movement at low metabolic cost. Force development and ATP utilisation during fixed-end contractions are typically five times slower in slow than fast muscles from the same species. Mechanical measurements previously suggested that more myosins are attached to thin filaments during contraction of slow muscle, which seems incompatible with its high efficiency. We therefore used small-angle X-ray diffraction to provide a structural estimate of the fraction of myosins attached to thin filaments in slow muscle. X-ray signals associated with myosin binding to actin indicate that only about 10% of myosin motors are actin-bound during fixed-end tetani of rat soleus slow muscles, compared with about 25% in mouse EDL fast muscle. Moreover, X-ray signals associated with the helical organisation of OFF myosin motors in the thick filaments show that about 70% of myosin motors remain in the OFF conformation during tetanic contraction of slow muscle, compared with only 30% in fast muscle. The much slower force development in soleus muscle also allowed clear separation of early structural changes in thick filaments on activation, some of which are distinct from those reported previously in fast muscles. Moreover, the early structural changes in soleus muscle have about the same amplitude in a twitch and a tetanus, suggesting that they are triggered by thin filament activation rather than thick filament stress, and implying a fast signalling pathway between thin and thick filaments. The interaction between myosin motors and actin filaments in slow skeletal muscles maintain posture and produce graded movement at low metabolic cost. Mechanical studies have suggested that more myosins are attached to actin filaments in slow than in fast muscle, but this seems incompatible with its high efficiency. We used X-ray diffraction to show that there are fewer myosin motors attached to actin in slow muscle than in fast muscle because more motors are sequestered on the myosin filament. The slower force development in slow muscle also allowed us to isolate and characterise fast changes in myosin motor conformation associated with activation of the actin filaments. The results reveal a distinct pathway of inter-filament signalling in slow muscle that could help the development of novel therapies for muscle weakness.