It is thought that fish generate the power needed for steady swimming with their anterior musculature, whereas the posterior musculature only transmits forces to the tail and does negative work. Isolated red muscle bundles driven through the length changes and stimulation pattern that muscles normally undergo during steady swimming showed the opposite pattern. Most of the power for swimming came from muscle in the posterior region of the fish, and relatively little came from the anterior musculature. In addition, the contractile properties of the muscle along the length of the fish are significantly adapted to enhance power generation.
Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.
To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 m⅐s ؊1 at 22°C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.
Insects, as a group, have been remarkably successful in adapting to a great range of physical and biological environments, in large part because of their ability to fly. The evolution of flight in small insects was accompanied by striking adaptations of the thoracic musculature that enabled very high wing beat frequencies. At the cellular and protein filament level, a stretch activation mechanism evolved that allowed high-oscillatory work to be achieved at very high frequencies as contraction and nerve stimulus became asynchronous. At the molecular level, critical adaptations occurred within the motor protein myosin II, because its elementary interactions with actin set the speed of sarcomere contraction. Here, we show that the key myosin enzymatic adaptations required for powering the very fast flight muscles in the fruit fly Drosophila melanogaster include the highest measured detachment rate of myosin from actin (forward rate constant, 3,698 s ؊1 ), an exceptionally weak affinity of MgATP for myosin (association constant, 0.2 mM ؊1 ), and a unique rate-limiting step in the cross-bridge cycle at the point of inorganic phosphate release. The latter adaptations are constraints imposed by the overriding requirement for exceptionally fast release of the hydrolytic product MgADP. Otherwise, as in Drosophila embryonic muscle and other slow muscle types, a step associated with MgADP release limits muscle contraction speed by delaying the detachment of myosin from actin.cross-bridge cycle ͉ Drosophila ͉ kinetics ͉ myosin I n Drosophila melanogaster two sets of antagonistic, asynchronous flight muscles oscillate at Ϸ200 beats per second, powering the wings indirectly by deforming the thoracic cuticle into which the muscles and wings insert. Although the myofibrillar basis of oscillatory work and power production is known (1-5), the molecular adaptations that allow the indirect flight muscles (IFM) to operate at very high frequencies are less well understood. In muscles of slow-to-moderate speed, muscle velocity is thought to be limited by prolonging the time myosin spends strongly bound to actin before detachment (6, 7). The prolongation is essential for coupling enzyme chemical kinetics, which normally occur rapidly, to the slower movements of the sarcomere during normal muscle function. In most vertebrate striated muscle types, the comparatively slow release of MgADP (one of the products of MgATP hydrolysis) is thought to be the ratelimiting step (8-13). However, recent studies suggest MgADP release may not be rate limiting for faster muscle types (14-16). If so, we reasoned that a shift in rate-limiting step to another part of the cross-bridge cycle should be most readily apparent in working indirect insect flight muscle, the fastest known muscle type.We had directly shown that myosin isoforms determine Drosophila IFM speed by using genetic engineering methods to substitute a relatively slow embryonic myosin (EMB) for the native fast myosin (IFI) in the IFM (Fig. 1A Inset) (17,18). This substitution transformed the ...
The genetic advantages of Drosophila make it a very appealing choice for investigating muscle development, muscle physiology and muscle protein structure and function. To take full advantage of this model organism, it has been vital to develop isolated Drosophila muscle preparations that can be mechanically evaluated. We describe techniques to isolate, prepare and mechanically analyze skinned muscle fibers from two Drosophila muscle types, the indirect flight muscle and the jump muscle. The function of the indirect flight muscle is similar to vertebrate cardiac muscle, to generate power in an oscillatory manner. The indirect flight muscle is ideal for evaluating the influence of protein mutations on muscle and cross-bridge stiffness, oscillatory power, and deriving cross-bridge rate constants. Jump muscle physiology and structure are more similar to skeletal vertebrate muscle than indirect flight muscle, and it is ideal for measuring maximum shortening velocity, force-velocity characteristics and steady-state power generation.
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