The contractile and regulatory proteins of insect flight muscle.
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ABSTRACT:
SUBMITTER: Bullard B
PROVIDER: S-EPMC1165820 | biostudies-other | 1973 Oct
REPOSITORIES: biostudies-other
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Project description:To assess the ability of the thin-filament regulatory system to control each stretch-activation (SA) event in the fast beating of asynchronous insect flight muscle (IFM), we obtained fast (3.4 ms/frame) and semistatic (> or = 50 ms) x-ray diffraction recordings for IFM fibers from bumblebees (beating at 170 Hz) and compared the results with those acquired in giant waterbugs (20-30 Hz) and crane flies (40 Hz, semistatic only). In contrast to the well-documented large SA force of waterbug IFMs, the SA force of bumblebee and crane fly IFMs was small compared to their large isometric force. In semistatic recordings, step-stretched bumblebee and crane fly IFMs showed smaller net SA-associated intensity changes in reflections that report myosin attachment to actin and tropomyosin movement toward its activating position. However, fast recordings on bumblebee IFMs showed a fast and large temporary reversal of intensities in these reflections, suggesting that the myosin heads supporting isometric force are dynamically replaced by SA-supporting heads, and that tropomyosin moves to and back from its inactivating position in milliseconds. In waterbug IFMs, the fast temporary reversal of intensities was not obvious. The observed rates of the attachment/detachment of myosin heads and the motion of tropomyosin are fast enough for the thin-filament regulatory system to control each SA event in fast-beating insects.
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Project description:Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.
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Project description:Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force-extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F(u) approximately 50-150 pN) than Ig-domains (F(u) approximately 150-250 pN). Among Ig domains in Sls/kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s(-1) (25 degrees C)] and even under high forces (15-30 pN). Temperature affected the unfolding forces with a Q(10) of 1.3, whereas the refolding speed had a Q(10) of 2-3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism.
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Project description: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.
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Project description:Both insect flight muscle and cardiac muscle contract rhythmically, but the way in which repetitive contractions are controlled is different in the two types of muscle. We have compared the flight muscle of the water bug, Lethocerus, with cardiac muscle. Both have relatively high resting elasticity and are activated by an increase in sarcomere length or a quick stretch. The larger response of flight muscle is attributed to the highly ordered lattice of thick and thin filaments and to an isoform of troponin C that has no exchangeable Ca2+-binding site. The Ca2+ sensitivity of cardiac muscle and flight muscle can be manipulated so that cardiac muscle responds to Ca2+ like flight muscle, and flight muscle responds like cardiac muscle, showing the malleability of regulation. The interactions of the subunits in flight muscle troponin are described; a model of the complex, using the structure of cardiac troponin as a template, shows an overall similarity of cardiac and flight muscle troponin complexes. The dual regulation by thick and thin filaments in skeletal and cardiac muscle is thought to operate in flight muscle. The structure of inhibited myosin heads folded back on the thick filament in relaxed Lethocerus fibres has not been seen in other species and may be an adaptation to the rapid contractions of flight muscle. A scheme for regulation by thick and thin filaments during oscillatory contraction is described. Cardiac and flight muscle have much in common, but the differing mechanical requirements mean that regulation by both thick and thin filaments is adapted to the particular muscle.
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Project description:The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5-6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ~40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ~98% of strong-binding acto-myosin attachments present after a length perturbation are confined to "target zones" of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle.
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