Sub-Millisecond Time-Resolved 2-Dimensional X-Ray Diffraction Recording from Stretch-Activated Flight Muscle from Bumblebee

Abstract

We have previously recorded the responses of X-ray reflections from bumblebee flight muscle (IFM) fibers to step length changes at a 3.4 ms time resolution (Iwamoto et al., Biophys. J., 2010). This study showed that, upon stretch, the myosin heads supporting isometric force detached, and were dynamically replaced by new myosin heads recruited by stretch activation (SA). At the same time, tropomyosin molecules (TM) temporarily moved back to its inactivating positions. These motions of proteins were very fast, and clearly the time resolution of the 3-CCD detector (640 x 480 pixels, 3.4 ms/full-frame) was insufficient. Here we used a new high-speed CMOS detector (1024 x 1024 pixels, up to 0.2 ms/full-frame) and repeated time-resolved measurements with a 0.5-ms time resolution. Unlike CCD detectors, the CMOS detector does not have to transmit charges serially, enabling a fast readout. In addition, we have improved the experimental design so that the IFM fibers tolerate higher X-ray dose (thus higher signal). Mitochondrial remnants, a major diffusion barrier for substances, were thoroughly removed to ensure sufficient ATP supply. After these improvements, the analysis from fully Ca-activated fibers showed that the stretch-induced detachment of myosin heads supporting isometric force is complete within 1 ms after completion of a 1-ms stretch, as shown by the reciprocal intensity changes of troponin reflections. The reattachment of myosin heads was slightly faster than the rise of SA force, and the number of attached heads started to decrease while the force was still rising. The movement of tropomyosin was perfectly synchronized with myosin detachment/reattachment, making it likely that tropomyosin and wings are beating synchronously in insects with “asynchronous” IFM. However, signals that trigger SA were not detected in the tropomyosin reflection even with this time resolution.