Three-dimensional Structure of Nucleotide-bearing Crossbridgesin Situ: Oblique Section Reconstruction of Insect Flight Muscle in AMPPNP at 23°C

Winkler, Hanspeter; Reedy, Mary C.; Reedy, Michael K.; Tregear, R. T.; Taylor, Kenneth A.

doi:10.1006/jmbi.1996.0642

Cited by 13 publications

(24 citation statements)

References 44 publications

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“…Two-headed “lead” cross-bridges (leading toward the M-line) occupy the four M-ward subunits (H–K) and the usually single-headed “rear” cross-bridges are bound to the most Z-ward pair (N, O) [55]. The extensive deformation of rigor rear bridges and their complete absence in AMPPNP [27], [56], suggests that the high actin affinity of nucleotide-free myosin extends the limit of acceptable actin azimuth.…”

Section: Discussionmentioning

confidence: 99%

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Reedy

et al. 2010

PLoS ONE

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BackgroundIsometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.MethodologyWe used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.ConclusionWe present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

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Section: Discussionmentioning

confidence: 99%

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Reedy

et al. 2010

PLoS ONE

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“…In addition, the solvent contains ATP which would preclude these being rigor-like cross-bridges, at least at these frequencies. Finally, on addition of AMPPNP, a non-hydrolyzable analog of ATP to rigor IFM, rear bridges detach and are not seen at all in averaged images (Winkler et al, 1996) and with very much less frequency in electron tomograms (Schmitz et al, 1996). …”

Section: Discussionmentioning

confidence: 99%

“…X-ray diffraction, fluorescent and paramagnetic probes, fiber mechanics and protein biochemistry produce information on the average state of the ensemble within the sample. Spatial averaging methods previously used for 3-D imaging of muscle (Taylor et al, 1984; Taylor et al, 1989b; Taylor et al, 1989a; Taylor et al, 1993; Winkler et al, 1996) have the same limitation. Single molecule methods, which must by necessity be applied to samples outside of the well ordered filament lattice of muscle, lose information on any effects imposed by organization of the lattice.…”

Section: Introductionmentioning

confidence: 99%

Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle

Reedy

et al. 2009

Journal of Structural Biology

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During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different kinetic steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze-substituted, Araldite embedded, thin sectioned and stained, to obtain 3-D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin-myosin attachments.

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“…These changes were explained either as arising from a change in the nature, but not the number, of the cross-bridges (Barrington-Leigh et al, 1973;Goody et al, 1975Goody et al, , 1976Beinbrech et al, 1976;Marston et al, 1976Marston et al, , 1979 or from a change in the activity of previously unattached cross-bridges (Wray, 1984). Later work showed that these interpretations were incorrect (Reedy et al, 1983a(Reedy et al, , 1987(Reedy et al, , 1988Tregear et al, 1990;Biosca et al, 1990;Schmitz et al, 1996;Winkler et al, 1996). AMPPNP actually causes the release of the rear cross-bridges while inducing only small changes in shape and attachment angle of the lead ones (middle top and bottom panels, Fig.…”

Section: Actin-myosin Interaction and Force Generationmentioning

confidence: 99%

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

Hooper

Hobbs

Thuma

2008

Progress in Neurobiology

126

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This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5 nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vetebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca ++ binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved.

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Three-dimensional Structure of Nucleotide-bearing Crossbridgesin Situ: Oblique Section Reconstruction of Insect Flight Muscle in AMPPNP at 23°C

Cited by 13 publications

References 44 publications

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

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