scholarly journals Tomographic Three-dimensional Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol

1997 ◽  
Vol 139 (3) ◽  
pp. 695-707 ◽  
Author(s):  
Holger Schmitz ◽  
Mary C. Reedy ◽  
Michael K. Reedy ◽  
Richard T. Tregear ◽  
Kenneth A. Taylor
Keyword(s):  
Ethylene Glycol ◽  
Flight Muscle ◽  
Insect Flight ◽  
Three Dimensional ◽  
Thick Filament ◽  
Power Stroke ◽  
Insect Flight Muscle ◽  
Target Zone ◽  
Dimensional Reconstruction ◽  
Myosin Subfragment 1

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5′-[β′γ- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension (“glycol-stiff state”). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of “glycol-stiff” sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90° axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45° angled rigor lead bridges. These 90° “target zone” bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These “nontarget zone” bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90° target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only ∼25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90° target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections

Nature ◽  
1984 ◽  
Vol 310 (5975) ◽  
pp. 285-291 ◽  
Author(s):  
Kenneth A. Taylor ◽  
Mary C. Reedy ◽  
Leonidas Córdova ◽  
Michael K. Reedy
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Three Dimensional ◽  
Three Dimensional Reconstruction ◽  
Insect Flight Muscle ◽  
Thin Sections ◽  
Dimensional Reconstruction

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

1989 ◽  
Vol 109 (3) ◽  
pp. 1103-1123 ◽  
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.

scholarly journals Binding of myosin subfragment 1 to glycerinated insect flight muscle in the rigor state

1985 ◽  
Vol 47 (2) ◽  
pp. 151-169 ◽  
Author(s):  
R.S. Goody ◽  
M.C. Reedy ◽  
W. Hofmann ◽  
K.C. Holmes ◽  
M.K. Reedy
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Insect Flight Muscle ◽  
Rigor State ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

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

1989 ◽  
Vol 109 (3) ◽  
pp. 1085-1102 ◽  
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.

Modification of crossbridge states by ethylene glycol in insect flight muscle

1984 ◽  
Vol 5 (1) ◽  
pp. 81-96 ◽  
Author(s):  
M. L. Clarke ◽  
C. D. Rodger ◽  
R. T. Tregear
Keyword(s):  
Ethylene Glycol ◽  
Flight Muscle ◽  
Insect Flight ◽  
Insect Flight Muscle

Three-dimensional image reconstruction of rigor crossbridges in insect flight muscle (IFM)

1983 ◽  
Vol 41 ◽  
pp. 454-455
Author(s):  
K. Taylor ◽  
L. Cordova ◽  
M.C. Reedy ◽  
M.K. Reedy
Keyword(s):  
Fourier Transforms ◽  
Flight Muscle ◽  
Insect Flight ◽  
Three Dimensional ◽  
Image Data ◽  
Insect Flight Muscle ◽  
Single Filament ◽  
Thin Filaments ◽  
Filament Axis ◽  
Axis Parallel

Dorsal longitudinal IFM from the water bug Lethocerus indicus, was rigorized by glycerination. Fiber bundle 958 retained a good rigor x-ray diffraction pattern after a 5 step fixation embedding sequence which included glutaraldehyde-tannic acid fixation, OsO4-UrAc staining, ethanol dehydration and Araldite embedding.The MYAC single filament layer of insect flight muscle contains alternating thick and thin filaments and is found isolated within longitudinal sections about 250Å thick. For our 3-D reconstruction we selected a MYAC region which appeared homogenous (both sides equal) over nearly 3/4 of a half sarcomere. Two tilt series ranging through ±60° of tilt in 10° steps were obtained, with tilt axis parallel to the filament axis in one and perpendicular in the other. Fourier transforms were calculated from 256x256 point arrays.The tilt data were combined using procedures similar to those used for 2-D crystalline arrays except that we obtain both amplitudes and phases from the image data.

Electron Tomography of Swollen Rigor Fibers of Insect Flight Muscle Reveals the S2 Fragment of Myosin

2001 ◽  
Vol 7 (S2) ◽  
pp. 98-99
Author(s):  
Yunxun Wang ◽  
Hanspeter Winkler ◽  
Michael K. Reedy ◽  
Mary C. Reedy ◽  
Kenneth A. Taylor
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Electron Tomography ◽  
Coiled Coil ◽  
Thick Filament ◽  
Insect Flight Muscle ◽  
Coil Structure ◽  
Close Proximity ◽  
Filament Spacing ◽  
First Time

We have used electron tomography of rigorized fibers of insect flight muscle swollen by immersion in dilute buffer (†1mM MOPS, pH 6.8), to reveal the 3-D arrangement of the myosin S2 fragment for the first time. S2 is an α-helical coiled-coil structure that connects the myosin head to the thick filament backbone. It is usually invisible in electron micrographs because of its close proximity to the backbone. The increase in inter-thick filament spacing from 45 nm to 55 nm pulls the S2 away from the filament backbone making it easier to identify. A total of 4 tomograms have been calculated by our standard method which involves collecting images using non-uniformly spaced tilt angles, cross correlation alignment, area matching and Whittaker-Shannon interpolation of the 3-D transform. A projection of one of these tomograms is shown in Figure 1. in raw tomograms S2 appears as a rod-like structure that originates at the thick filament surface to connect to both lead and rear crossbridges of rigor. S2 segments are particularly well resolved at the ends of the thick filament (Fig. 2), where in some cases it appears that the myosin subfilaments are separated from the backbone. Both the azimuthal direction and axial angle of S2 are variable.

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

1996 ◽  
Vol 264 (2) ◽  
pp. 302-322 ◽  
Author(s):  
Hanspeter Winkler ◽  
Mary C. Reedy ◽  
Michael K. Reedy ◽  
Richard Tregear ◽  
Kenneth A. Taylor
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Insect Flight Muscle ◽  
Oblique Section ◽  
Three Dimensional Structure
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