Abstract
Small insects drive their flight muscle at frequencies up to 1,000 Hz. This remarkable ability owes to the mechanism of stretch activation. However, it remains unknown as to what sarcomeric component senses the stretch and triggers the following force generation. Here we show that the earliest structural change after a step stretch is reflected in the blinking of the 111 and 201 reflections, as observed in the fast X-ray diffraction recording from isolated bumblebee flight muscle fibers. The same signal has also been observed in live bumblebee. We demonstrate that (1) the signal responds almost concomitantly to a quick step stretch, (2) the signal grows with increasing calcium levels as the stretch-activated force does, and (3) a full 3-dimensional model demonstrates that the signal is maximized when objects having a 38.7-nm actin periodicity travel by ~20 nm along the filament axis. This is the expected displacement if myosin heads are loosely associated with actin target zones (where actin monomers are favorably oriented), and are dragged by a 1.3% stretch, which effectively causes stretch-induced activation. These results support and strengthen our proposal that the myosin head itself acts as the stretch sensor, after calcium-induced association with actin in a low-force form.
Highlights
A stretch, and they occur ahead of the changes of other reflections that parallel the stretch activation (SA) force generation
Responses of X-ray reflections to stretch of fully calcium-activated skinned insect flight muscle (IFM) fibers from bumblebee
An array of skinned bumblebee IFM fibers were mounted in a specimen chamber, and were step stretched after full calcium activation, and time-resolved X-ray recordings were done at the high-flux BL40XU beamline of SPring-8 as were done previously[10]
Summary
A stretch, and they occur ahead of the changes of other reflections that parallel the SA force generation. Ex-vivo observations may not be identical to what occurs in vivo, the skinned fiber preparations allow precise control of experimental conditions, and much smaller background X-ray scattering allows analyses of weaker reflections In these experiments, X-ray diffraction patterns were taken at 2,000 frames/s instead of 5,000 frames/s for live bumblebees, but the lower experimental temperature (20° vs ~42° in the thorax of a live bumblebee) gives enough time resolution to record responses to 1-ms step stretches. We further expand the original 2-D model[12] to a full 3-D model, to explore possible structural changes of contractile proteins in the 3-D space of myofilament lattice This 3-D model demonstrates that the observed changes of the 111 and 201 reflections are satisfactorily explained, if myosin heads are pulled along the filament axis while they are loosely bound to actin. These results provide further support for the idea that the SA-triggering mechanism is built into myosin itself
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