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.

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.

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.

A new 1.4 nm gold-Fab probe

1991 ◽  
Vol 49 ◽  
pp. 284-285
Author(s):  
James F. Hainfeld ◽  
Frederic R. Fumya ◽  
Richard D. Powell
Keyword(s):  
High Resolution ◽  
Gold Particle ◽  
Colloidal Gold ◽  
Size Range ◽  
Life Sciences ◽  
Major Advance ◽  
Trial And Error ◽  
Fab Fragment ◽  
Gold Particles ◽  
Error Testing

A major advance in high resolution EM immunoprobes has recently been achieved: The smallest gold particles easily seen directly in the TEM have been coupled to Fab fragments thus making them the smallest gold-antibody probe commercially available.The gold particle, NanogoldTM, is 1.4 nm in diameter with a very controlled size range, ± 10% (Fig. 1). This is in sharp contrast to other small gold preparations, such as Auroprobe One (Janssen Life Sciences) which actually ranges from 1-3 nm.The Fab conjugate (Fig. 2) has close to one gold particle per Fab fragment. This again is different from other gold-IgG probes that have 0.2-10 gold particles per IgG. Another difference is that the Nanogold-Fab conjugates are separate molecules in solution rather than the often extensive aggregation of other colloidal gold-IgG preparations. The problems of ratio of gold particles to antibody and aggregation in conventional colloidal conjugates were shown to be controllable by careful trial and error testing.

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

1980 ◽  
Vol 207 (1166) ◽  
pp. 13-33 ◽  
Keyword(s):  
Thin Filament ◽  
Flight Muscle ◽  
Insect Flight ◽  
Insect Flight Muscle ◽  
X Ray Diffraction ◽  
Thin Filaments ◽  
Model Transform ◽  
Cross Bridges ◽  
The Fourier Transform ◽  
Symmetry Operations

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.

scholarly journals The length–tension curve in muscle depends on lattice spacing

2013 ◽  
Vol 280 (1766) ◽  
pp. 20130697 ◽  
Author(s):  
C. David Williams ◽  
Mary K. Salcedo ◽  
Thomas C. Irving ◽  
Michael Regnier ◽  
Thomas L. Daniel
Keyword(s):  
Lattice Spacing ◽  
Striated Muscle ◽  
Flight Muscle ◽  
Insect Flight ◽  
Radial Distance ◽  
Muscle Length ◽  
Insect Flight Muscle ◽  
Myosin Filaments ◽  
New Models ◽  
Tension Curve

Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle's force–length dependence.

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