Interpretation of the low angle X-ray diffraction from insect flight muscle in rigor

The structure of insect flight muscle is formally described in terms of actin-based cross-bridges upon which successive symmetry operations are performed, in combination with a modulation function. The Fourier transform of the structure is generated by means of these steps. The model transform is fitted to the observed diffraction pattern from insect flight muscle in rigor and the position of the rigor cross-bridges deduced; they are found to lie across the long helix of actin monomers and to project away from the thin filament. The cross-bridges interact with approximately one-third of the actin monomers, and show a strong preference for a particular orientation between the thick and thin filaments.

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Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.1085 ◽

1989 ◽

Vol 109 (3) ◽

pp. 1085-1102 ◽

Cited By ~ 22

Author(s):

K A Taylor ◽

M C Reedy ◽

L Córdova ◽

M K Reedy

Keyword(s):

Thin Filament ◽

Flight Muscle ◽

Insect Flight ◽

Three Dimensional ◽

Bridge Structure ◽

Insect Flight Muscle ◽

Single Filament ◽

Cross Bridge ◽

Cross Bridges

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.

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X-Ray Diffraction Indicates That Active Cross-Bridges Bind to Actin Target Zones in Insect Flight Muscle

Biophysical Journal ◽

10.1016/s0006-3495(98)77856-7 ◽

1998 ◽

Vol 74 (3) ◽

pp. 1439-1451 ◽

Cited By ~ 47

Author(s):

R.T. Tregear ◽

R.J. Edwards ◽

T.C. Irving ◽

K.J.V. Poole ◽

M.C. Reedy ◽

...

Keyword(s):

Flight Muscle ◽

Insect Flight ◽

Insect Flight Muscle ◽

X Ray Diffraction ◽

Target Zones ◽

X Ray ◽

Cross Bridges

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Interplay between passive tension and strong and weak binding cross-bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal.

The Journal of General Physiology ◽

10.1085/jgp.101.2.235 ◽

1993 ◽

Vol 101 (2) ◽

pp. 235-270 ◽

Cited By ~ 59

Author(s):

H L Granzier ◽

K Wang

Keyword(s):

Mechanical Properties ◽

Ionic Strength ◽

Thin Filament ◽

Flight Muscle ◽

Insect Flight ◽

Passive Tension ◽

Passive Stiffness ◽

Insect Flight Muscle ◽

Weak Binding ◽

Cross Bridges

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.

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Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.1103 ◽

1989 ◽

Vol 109 (3) ◽

pp. 1103-1123 ◽

Cited By ~ 17

Author(s):

K A Taylor ◽

M C Reedy ◽

L Córdova ◽

M K Reedy

Keyword(s):

Thin Filament ◽

Flight Muscle ◽

Insect Flight ◽

Three Dimensional ◽

Leading Edge ◽

Power Stroke ◽

Insect Flight Muscle ◽

Single Filament ◽

Filament Axis ◽

Cross Bridges

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.

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1P142 X-ray diffraction from insect flight muscle with exchanged contractile proteins(10. Muscle,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Seibutsu Butsuri ◽

10.2142/biophys.54.s164_4 ◽

2014 ◽

Vol 54 (supplement1-2) ◽

pp. S164

Author(s):

Hiroyuki Iwamoto ◽

Naoto Yagi

Keyword(s):

Annual Meeting ◽

Flight Muscle ◽

Insect Flight ◽

Contractile Proteins ◽

Insect Flight Muscle ◽

X Ray Diffraction ◽

X Ray ◽

Biophysical Society

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A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling

Journal of Cell Science ◽

10.1242/jcs.101.3.503 ◽

1992 ◽

Vol 101 (3) ◽

pp. 503-508

Author(s):

R. Newman ◽

G.W. Butcher ◽

B. Bullard ◽

K.R. Leonard

Keyword(s):

Gold Particle ◽

Colloidal Gold ◽

Striated Muscle ◽

Flight Muscle ◽

Insect Flight ◽

Insect Flight Muscle ◽

Fab Fragment ◽

Thin Filaments ◽

Gold Particles ◽

Almost All

Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosin-troponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately +/− 4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.

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