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• Articles by M. Anson
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• Passive Stiffness in Drosophila Indirect Flight Muscle Reduc...

  Passive Stiffness in Drosophila Indirect Flight Muscle Reduced by Disrupting Paramyosin Phosphorylation, but Not by Embryonic Myosin S2 Hinge Substitution Biophysical Journal, Volume 91, Issue 12, 15 December 2006, Pages 4500-4506 Yudong Hao, Mark S. Miller, Douglas M. Swank, Hongjun Liu, Sanford I. Bernstein, David W. Maughan and Gerald H. Pollack Abstract High passive stiffness is one of the characteristic properties of the asynchronous indirect flight muscle (IFM) found in many insects like . To evaluate the effects of two thick filament protein domains on passive sarcomeric stiffness, and to investigate their correlation with IFM function, we used microfabricated cantilevers and a high resolution imaging system to study the passive IFM myofibril stiffness of two groups of transgenic lines. One group (hinge-switch mutants) had a portion of the endogenous S2 hinge region replaced by an embryonic version; the other group (paramyosin mutants) had one or more putative phosphorylation sites near the N-terminus of paramyosin disabled. Both transgenic groups showed severely compromised flight ability. In this study, we found no difference (compared to the control) in passive elastic modulus in the hinge-switch group, but a 15% reduction in the paramyosin mutants. All results were corroborated by muscle fiber mechanics experiments performed on the same lines. The fact that myofibril elasticity is unaffected by hinge switching implies alternative S2 hinges do not critically affect passive sarcomere stiffness. In contrast, the mechanical defects observed upon disrupting paramyosin phosphorylation sites in suggests that paramyosin phosphorylation is important for maintaining high passive stiffness in IFM myofibrils, probably by affecting paramyosin’s interaction with other sarcomeric proteins. Abstract | Full Text | PDF (248 kb)

• Muscle Mechanics & Ultrastructure - I

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• Aging Enhances Indirect Flight Muscle Fiber Performance yet ...

  Aging Enhances Indirect Flight Muscle Fiber Performance yet Decreases Flight Ability in Drosophila Biophysical Journal, Volume 95, Issue 5, 1 September 2008, Pages 2391-2401 Mark S. Miller, Panagiotis Lekkas, Joan M. Braddock, Gerrie P. Farman, Bryan A. Ballif, Thomas C. Irving, David W. Maughan and Jim O. Vigoreaux Abstract We investigated the effects of aging on indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance. Abstract | Full Text | PDF (996 kb)

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Abstract

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.