Steric blocking mechanism explains stretch activation in insect flight muscle.

O ver 60 y have elapsed since John Pringle (1) described a remarkable property of insect flight muscles: If you stretch them, after short a delay, they exert more force. This explains how insects beat their wings. The flight muscles are organized in antagonistic pairs coupled to the wing. The system is a free-running oscillator: Each time one muscle contracts, it stretches the opposing muscle, which then contracts. How does this come about? Muscle works by myosin cross-bridges interacting with actin to move the actin filaments past the myosin filaments (reviewed in 2). Muscle movement is inhibited if the myosin crossbridges cannot bind to the actin filaments. In skeletal muscle, the release of Ca under nervous control results in the movement of tropomyosin molecules across the surface of the actin filaments to uncover the myosin-binding sites on actin (3–5)—the steric blocking model. Flight muscles are switched on by release of Ca into the sarcoplasm; however, in insect flight muscles, Ca alone is not enough to free up the myosin-binding sites—you need stretch as well. The paper in PNAS by Perz-Edwards et al. (6) shows that the stretch activation of insect flight muscle uses the same steric blocking model for controlling the actin-myosin interaction that was found in skeletal muscle. In the case of insect flight muscles, stretching the muscle causes the tropomyosin to move across the surface of actin to free up the myosin-binding sites. Fig. 1 shows the actin filament with the associated proteins tropomyosin and troponin [based on the article by Poole et al. (7)]. The troponin complex is a trimer of TnT, TnI, and TnC proteins. TnT binds to tropomyosin. TnI anchors troponintropomyosin to actin. TnC, a standard EFhand protein that binds Ca, controls this affinity. There are two flight muscle isoforms of TnC, one of which enables stretch activation (8, 9). Tropomyosin is a long double–α-helical coiled coil that lies along the surface of the actin filament. The tropomyosin molecules aggregate end to end, and troponin binds near the tropomyosin junctions. Under control of the troponin complex, three states of tropomyosin can be characterized (10): “blocked,” “closed,” and “open” (Fig. 2). In the absence of Ca, the troponin complex holds tropomyosin in the blocked position. Ca binding enables TnC to detach TnI from actin and allows the tropomyosin to move. In Lethocerus and all other flight muscles, TnC binding of calcium is necessary but not sufficient for activation: Tropomyosin is held in the blocked position until the muscle is stretched. Only then does tropomyosin move to uncover the myosin cross-bridge– binding sites on actin. The myosin crossbridges can bind with a short delay that is characteristic of stretch activation. Time-resolved X-ray fiber diffraction allows observation of the molecular movements underlying muscle contraction. Indeed, time-resolved measurements and measurements in different physiological states have contributed substantially to our present understanding of the molecular mechanism of muscle contraction (11–13). One early observation concerned tropomyosin (3–5). The second actin layer-line of the diffraction pattern is responsive to the four-foldedness of the actin filament. On electrical stimulation of an intact frog muscle, the tropomyosin moves from blocked to open. This movement gives the actin filament a much more fourfold appearance. As a result, when the tropomyosin is in the open position, there is a peak (so-called “J4 peak”) on the second actin layer-line that vanishes at the end of stimulation when tropomyosin returns to the blocked position. The giant water bug Lethocerus provides a convenient model system for studying flight muscles (14). If Lethocerus flight muscle fibers, treated so as to remove the cell membranes, are mounted in a suitable machine, they will oscillate for hours if fed with ATP. Observation of large ATP-induced intensity changes in the 14.5-nm meridional X-ray reflection from insect flight muscle showed the myosin crossbridge to be capable of taking up two different orientations (15). Moving between these two states drives contraction (16). Spurred on by this early discovery, Mike Reedy (and, later, Mary Reedy and their collaborators) devoted themselves to trying to understand the structure and function of insect flight muscle. Their tools were electron microscopy for static structures and time-resolved X-ray fiber Fig. 1. Thin filament [based on the article by Poole et al. (7)]. Actin monomers form a helical filament (space-filling models, shown in blue). Tropomyosin molecules (shown in cartoon representation in red and yellow) are long two-chained α-helical coiled coils that curl round the surface of the actin molecule. The tropomyosin molecules aggregate end to end. The troponin complexes (green), which are trimers (i.e., TnC, TnI, TnT), bind near each of the tropomyosin junctions and to the actin filament. In the absence of Ca, the troponin complex holds tropomyosin in the blocked position. The shaded area is shown in Fig. 2. Author’s contributions: K.C.H. wrote the paper.