Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit Skeletal Muscle Actin

Abstract

Stretch activation (SA, delayed rise of force after stretch) is a mechanism essential for the fast wing-beat of insects with asynchronous flight muscles. To determine which constituent protein(s) is (are) indispensable for SA, we have been conducting experiments to replace the endogenous actin of bumblebee flight muscle with rabbit skeletal muscle actin, and we have demonstrated that actin filaments can be regenerated as judged from X-ray diffraction patterns (2016 annual meeting). In this experiment, the endogenous actin is removed by gelsolin, and after this, rabbit G-actin is polymerized in situ. To prevent spontaneous contraction, a myosin inhibitor must be added to the solutions. For vertebrate skeletal and cardiac muscles, the preferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996). However, BDM is not an effective inhibitor for insect flight muscle. For this reason, we used blebbistatin instead, and the actin filaments were successfully restored. The problem is that the inhibition by blebbistatin is irreversible, and it is not reversed by extensive washout or irradiation with blue light. Here we used a high concentration of BDM (100 mM) and repeated the experiments. The actin filaments were restored, and unlike in the case of blebbistatin, actin-based layer line reflections were intensified after washout of BDM and ATP. This indicates that the ability of the endogenous myosin to form rigor complexes is not compromised by the use of BDM. We are currently trying to activate the flight muscle fibers prepared in this way.