Abstract

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.