Fusi, L. | Search bioRxiv

bioRxiv2025

Sub-sarcomeric regulation of thin and thick filaments in skeletal muscle myofibrils

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.

biophysics↗

bioRxiv2025

Distinct distributions of myosin motor conformations during contraction of slow and fast skeletal muscle

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. Key PointsO_LIThe interaction between myosin motors and actin filaments in slow skeletal muscles maintain posture and produce graded movement at low metabolic cost. C_LIO_LIMechanical 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. C_LIO_LIWe 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. C_LIO_LIThe 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. C_LIO_LIThe results reveal a distinct pathway of inter-filament signalling in slow muscle that could help the development of novel therapies for muscle weakness. C_LI

biophysics↗