Flight muscle-specific Pro-Ala-rich extension of troponin is important for maintaining the insect-type myofilament lattice integrity

Insect flight muscle (IFM) can oscillate at frequencies up to 1000Hz, owing to its capability of stretch activation (SA). It is a highly specialized form of cross striated muscles, and its peculiar features include the IFM-specific isoform of troponin-I (troponin-H or TnH) with an unusually long Pro-Ala-rich extension at the C-terminus. Although we have shown that this extension does not directly take part in SA, questions remain as to what its real role is and why it is expressed only in IFM. Here we explored the structural role of the extension, be comparing X-ray diffraction patterns and electron micrographs of bumblebee IFM fibers before and after enzymatic removal of the extension. The removal had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much stronger than the 1,1 reflection, but after removal, their intensities became almost equal (stronger 1,1 is a feature of vertebrate skeletal muscle). Electron micrographs revealed that a substantial fraction of the thin filaments showed a tendency to move towards the vertebrate position (the trigonal position between three thick filaments), while the rest of the thin filaments remained in their original insect position (midway between two neighboring thick filaments). Therefore, one of the roles of the extension is suggested to keep the filament lattice in the correct configuration for IFM. This insect-type lattice structure is preserved among IFMs from varied insect orders but not in body muscles, suggesting that the maintenance of this lattice structure is important for flight functions.