Regeneration of Actin Filaments in Actin-Extracted Bumblebee Flight Muscle Fibers as Probed by X-ray Diffraction

Abstract

Asynchronous flight muscle enables the insects to beat their wings at high frequencies not achievable by ordinary twitch-type muscles. To understand the mechanism of action of asynchronous flight muscle, it is essential to know which of the constituent contractile proteins (many of them are expressed as flight muscle-specific isoforms) is/are indispensable or replaceable. For this purpose, we tried to extract myosin from the flight muscle and replace it with that of vertebrate skeletal muscle, by increasing ionic strength. However, this procedure seemed to extract other proteins important for maintaining the integrity of the myofibril (2015 annual meeting). Alternatively, we have established a protocol to extract actin from bumblebee flight muscle fibers by using gelsolin, an actin-severing protein. After treatment with gelsolin, actin layer line reflections disappeared from the X-ray diffraction pattern, but reflections from myosin filaments and their lattice remained basically intact. The results show that gelsolin treatment is a method suitable for extracting actin and its associated proteins without sacrificing the structural integrity of the myofibril. As a next step, we are currently trying to introduce exogenous actin (from vertebrate skeletal muscle) and to see how the contractile properties of the flight muscle fibers are affected.