A C-terminal Truncation of Flightin Slows Actomyosin Cycling, Elevates Passive Tension, and Decreases Power Output in Drosophila Flight Muscle Fibers

Abstract

Flightin is a 182 amino acid myosin rod binding protein that is essential for thick filament stability and function in the indirect flight muscles (IFM) of Drosophila melanogaster. Flightin is not homologous to any known protein in vertebrates, but recent phylogenetic analyses revealed that flightin is widespread among insects and crustaceans. Amino acid sequence analysis suggests that flightin consists of three functional domains: a fast evolving and highly phosphorylated N-terminal domain (residues 1-83), a conserved central domain (residues 84-134) and a semi-conserved C-terminal domain (residues 135-182). To interrogate the function of the C-terminal domain, we created a transgenic Drosophila that expresses a truncated flightin, missing the last 43 residues (fln142t). These transgenic flies are unable to beat their wings, but their myofilament lattice and sarcomere structure appear normal compared to a flightin-null rescued control line (flnR). Active (pCa 5) and rigor isometric tensions in skinned IFM fibers were higher for fln142t versus flnR, which occurs due to a 2-fold increase in passive (pCa 8) isometric tension. Small amplitude sinusoidal perturbation analysis showed that fln142t fibers produced approximately 30% of the oscillatory work and power of flnR fibers. The fln142t also showed a decreased frequency of maximum work (123 Hz vs. 154 Hz) and power (139 Hz vs. 187 Hz) compared to flnR, suggesting slower myosin kinetics even though myosin attachment time (=0.5 ms) was unchanged. These results suggest that the C-terminus of flightin plays a limited role in thick filament integrity and normal sarcomere structure of the IFM, but is essential for maintaining the muscle's passive properties and tuning the kinetic properties during contraction.