Antiparallel dimerization of obscurin controls M-line assembly in Drosophila indirect flight muscle

The giant scaffold protein obscurin plays a crucial role in anchoring thick filaments at the muscle M-line. Here we show that Drosophila obscurin forms an antiparallel dimer at the M-line with the N-terminal SH3-DH-PH domain facing outwards toward the thin filaments, and the C-terminal kinase region involved in dimerization or oligomerization as well as myosin binding. This arrangement is supported by myosin co-immunoprecipitation, yeast two-hybrid, and super-resolution microscopy data, as well as Obscurin mutants specifically affecting the N- versus the C-terminus. While deletion of the N-terminal domains of obscurin predominantly affects peripheral thin filament length, loss of the C-terminal kinase region of obscurin disrupts early sarcomere assembly leading to various defects during sarcomere maturation, supporting its role in keeping thick filaments in register. In conclusion, we propose that obscurin forms a structural and elastic component organizing thick filaments with its C-terminal kinase region at the M-line, while connecting to thin filaments with its N-terminal domains.