Highlights d A key residue variation of fast skeletal myosin allows selective targeting d A rationally designed inhibitor (MPH-220) specifically blocks skeletal muscle d The inhibitor-bound myosin atomic structure reveals mechanism of selectivity d MPH-220 improves gait disorders in spastic animal model after brain injury Authors Ma ´te ´Gyimesi, A ´da ´m I. Horva ´th,

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog ( Rana esculenta ), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm −1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2′s allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2′s upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins. Stress fibers are contractile actomyosin bundles in non-muscle cells, playing fundamental roles in diverse biological functions 1-4. Stress fibers and stress fiber-like structures play central roles in cell division as key components of the cleavage furrow 5 , determine cancer cell motility during metastasis 6 , influence cancer cell growth 7 , and they have been demonstrated as crucial elements in neuronal plasticity including neurite outgrowth 4,8-11. These force-generating structures consist of actin filaments and different co-assemblies of non-muscle myosin 2 (NM2) isoforms 12,13. The two major stress fiber-forming myosin isoforms in human cells are NM2A and NM2B, that together build up mixed minifilaments of various length and thickness 14. Stress fibers may be categorized as central and peripheral, based on their subcellular localization. Central stress fibers include those localized ventrally, dorsally, in transverse arcs and those associated around the nucleus. Their main roles are in cell adhesion, motility, cell division and cell shape determination 15,16. Peripheral stress fibers are located at the edge of the cell. Laser nanosurgery experiments have shown that incision of peripheral stress fibers causes them to retract significantly, suggesting that these structures are under larger mechanical strain than their central counterparts 17. Resisting loads on NM2 heads have been shown to significantly decrease the rate of ADP release from actin-bound myosin heads, resulting in slower actomyosin dissociation 18-20. NM2 minifilaments are also activated or repressed by the phosphorylation or dephosphorylation of the myosin light chains (MLC), respectively 21-24. Rho-dependent kinase (ROCK) positively regulates...