The organisation of titin at the centre of the vertebrate striated muscle thick filament.

Abstract

Striated muscle sarcomeres operate by sliding of the regular arrays of interdigitating thin (actin) and thick (myosin) filaments. Thick filaments are held together at their centre by M-band bridges, maintaining sarcomere structure during contraction and relaxation. The giant sarcomeric protein titin is instrumental in maintaining order, since C-terminal truncations of titin lead to loss of A-band alignment and sarcomere failure. The C-terminal sequence of titin encodes a kinase domain, 10 Ig domains and linker sequences, and is associated with the bare zone at the centre of the sarcomere. Unlike the cross-bridge associated sequence, it does not show any correlation with the organisation of the thick filament bare zone and associated M-band bridges. The precise disposition of titin remains unclear, e.g. whether anti-parallel molecules overlap at centre of the filament. We used superresolution light microscopy with new titin antibodies to establish the position of several titin epitopes in the bare zone, together with key markers for the M-band proteins myomesin and M-protein. We show that the titin kinase domain is at the edge of the bare zone, about 70 nm from the M-band centre at the first myosin motor domains and may therefore account for the first of the accessory protein stripes in the A-band. SAXS data of two titin constructs covering several domains of the bare-zone associated sequence, one from the beginning including the kinase and the other from the end, suggest that they are extended structures but with compact linkers. Conversely, the long insert sequence between M-band Ig-domains 3 and 4 (Ig-162 and Ig-163) appears to be more extensive. Our combined data suggests that in the bare zone, titin molecules from each side of the thick filament overlap each other by the width of the M-band.