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.

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 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.

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

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.

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 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.

Structure of the insect flight muscle Z disk

1983 ◽  
Vol 41 ◽  
pp. 456-457
Author(s):  
Deatherage J.F. ◽  
Luke B. ◽  
Sainsbury G.M. ◽  
Bullard B.
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Hexagonal Lattice ◽  
Unit Cell ◽  
Insect Flight Muscle ◽  
Thin Filaments ◽  
Thin Sections ◽  
Carpenter Bee ◽  
Contour Maps ◽  
Fourier Filtering

The Z disk of striated muscle is a regular planar network of connections linking two sets of oppositely directed thin filaments from adjacent sarcomeres. Ashurst has inspected images of oblique thin sections through insect Z disks, and proposed that parallel overlapping filaments in the Z disk are arranged in hexagonal bundles of six, which alternating filaments enter from opposite sarcomeres. We have now analyzed images of thin-sectioned and negatively-stained Z disks using Fourier filtering and crystallographic image processing methods. Our findings confirm the basic model of Ashurst for the arrangement of filaments inside the Z disk. By evaluating contour maps of crystallographically averaged images at higher resolution, we have obtained additional information about the positions of the filaments and the connections linking them.We have examined Z disks of flight muscles of three orders of insect, Diptera (blowfly), Hemiptera (Lethocerus) and Hymenoptera (bumble and carpenter bee). Thin sections from Lethocerus whole muscle fibers and negatively-stained isolated Z disks from bumble bee were examined in most detail. The connectivities of thin filaments of one sarcomere through the Z disk to the adjacent sarcomere were traced on Fourier-filtered images of oblique thin-sections. These cut the myofibril at an angle off perpendicular, intersecting it on one side of the Z disk, passing through it, and exiting on the other side. The filaments in the Z disk lattice, and the connections between them, were examined on crystallographically averaged images of transverse sections through the Z disk, and of isolated, negatively-stained Z disks.The insect flight muscle Z disk is arranged on a hexagonal lattice with unit cell dimensions of about 465Å. Images of both thin- sectioned and negatively-stained Z disks diffract optically to the fifth or sixth order (about 75Å resolution). The projection symmetry of the Z disk is p3m. Contour maps of crystallographically averaged images of thin- sectioned and isolated Z disks show prominent features arranged in roughly hexagonal bundles of six. These features correspond to extensions of the thin filaments into the Z disk, three from each of the opposing sarcomeres. By analysis of oblique sections we have confirmed that the opposed thin filament lattices of the two sarcomeres meet in three-fold to six-fold register (that is, a third of a unit cell out of register along both crystallographic axes). The relative strengths of features in these images are consistent with long overlap of filaments from opposite sarcomeres inside the Z disk.

Sign in / Sign up

Export Citation Format

Share Document