Phosphorylation-dependent power output of transgenic flies: an integrated study

Dickinson, Michael H.; Hyatt, Christopher J.; Lehmann, Fritz-Olaf; Moore, Jeffrey R.; Reedy, Mary C.; Simcox, Amanda; Tohtong, Rutaiwan; Vigoreaux, Jim O.; Yamashita, Hiroshi; Maughan, David W.

doi:10.1016/s0006-3495(97)78338-3

Cited by 92 publications

(106 citation statements)

References 51 publications

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“…Aluminum T-clips were attached to each segment, yielding a preparation Ϸ0.6 mm long and Ϸ0.1 mm wide. The segment was transferred to a 30-l drop of glycerol-and detergent-free relaxing solution and mounted between a piezoelectric motor and strain gauge on a mechanical rig (25). The temperature was set at 15°C.…”

Section: Methodsmentioning

confidence: 99%

“…For each solution condition, the complex modulus of each fiber was fitted to a three-term equation (25,30) by following the Predicted shifts of apparent rate constants from calculations based on eight schemes with different rate-limiting steps (corresponding to numbers in the cross-bridge scheme in C). The details of the calculations are given in supporting information.…”

Section: Methodsmentioning

confidence: 99%

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An exceptionally fast actomyosin reaction powers insect flight muscle

Swank

Vishnudas

Maughan

2006

Proc. Natl. Acad. Sci. U.S.A.

115

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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 ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

An exceptionally fast actomyosin reaction powers insect flight muscle

Swank

Vishnudas

Maughan

2006

Proc. Natl. Acad. Sci. U.S.A.

115

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“…Fibers were immediately used for mechanical experiments or were transferred to storage solution (relaxing solution plus protease inhibitor mixture and 50% glycerol) at Ϫ20°C and used within a few days of dissection. Skinned fibers, in relaxing solution, were attached to aluminum T-clips at both ends and mounted to a force transducer and a length driver (23,41). The fiber was stretched until just taut (minimal stress), and the initial fiber length was measured.…”

Section: Methodsmentioning

confidence: 99%

“…To perform sinusoidal analysis, small-amplitude sinusoidal length changes (0.125% muscle length) were applied to the fiber at 47 frequencies (0.5-1,000 Hz) while measuring the force response (23). Length and force were normalized to determine strain (⌬L͞L) and tension (F͞CSA) by dividing the length change (⌬L) by total fiber length (L) and by dividing the force (F) by the fiber crosssectional area (CSA).…”

Section: Methodsmentioning

confidence: 99%

“…Phosphorylation is an important means of regulating thick filament proteins such as vertebrate myosin binding protein-C (21), vertebrate and invertebrate myosin regulatory light chain (22)(23)(24)(25), and Drosophila flightin (26,27). Paramyosin is phosphorylated in vivo (5,28) and can be phosphorylated in vitro (29)(30)(31)(32), specifically in the nonhelical N-terminal domain (32).…”

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confidence: 99%

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Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster

Liu

Miller

Swank

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and͞or stabilize thick filament interactions with proteins that contribute to fiber stiffness.contraction ͉ thick filament ͉ sarcomere ͉ mechanics ͉ insect P aramyosin is a major structural component of thick filaments in invertebrate muscle. The paramyosin to myosin ratio, which positively correlates with thick filament length and maximum active tension development (1), varies widely between different invertebrate phyla and even between different muscles within the same animal (2-5). For instance, this ratio is 1:34 in the fibrillar flight muscle of Drosophila melanogaster but is 1:6 in larval muscles (5). Sequence analysis of paramyosin isoforms isolated from different species reveal a rod-like molecule with a central ␣-helical region and two nonhelical terminal domains that can dimerize into a coiled-coil structure (6, 7). Like the myosin tail, amino acids of the paramyosin helical region have a periodic 28-residue positive and negative charge repeat distribution (6,8). This charge arrangement on the surfaces of paramyosin and the myosin tail may regulate the packing of these two proteins in the thick filament (6, 9, 10).Paramyosin is important for thick filament formation and for filament assembly into myofibrils (11,12). A currently accepted model of invertebrate thick filament assembly is that paramyosin molecules, together with other thick filament structur...

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