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Fig. 9. Electron microscopic observation on thick filaments labeled with
anti-twitchin D2 peptide antibody. (A) Electron micrographs of ABRM thick
filaments labeled with the anti-twitchin D2 peptide antibody and negatively
stained. Antibodies conjugated with gold particles, indicating localization of
twitchin, are distributed on the surface of the filaments at intervals (upper
panel) and at helical turns (lower panel). (B) Electron micrograph of a thick
filament treated with low angle rotary shadowing after negative staining. The
secondary antibody-conjugated gold particles are localized on globular
structures. (C) Stereo views of negatively stained thick filaments. Ultrathin
filaments, possibly representing twitchin molecules, expand longitudinally on
the thick filament as indicated by the white arrow. Arrowheads indicate
location of the antibody-conjugated gold particles. (D) Electron microscopic
observation of twitchin molecules by rotary shadowing. Twitchin molecule
(left) and after treatment with anti-twitchin kinase domain antibody (right).
Twitchin (0.06 mg ml–1) was reacted with the anti-twitchin
kinase domain antibody (Funabara et al.,
2001a) and mixed with 40% glycerol. This preparation, and a sample
of twitchin without antibody, were sprayed onto mica and subjected to rotary
shadowing using platinum and carbon as described above. (E) Models of the
parallel array of twitchin molecules (red) superimposed on the Bear-Selby net
pattern (Bear and Selby, 1956)
and relative to myosin head distribution (blue). Bars, 100 nm (A–C), 50
nm (D). The Bear-Selby net reflects the arrangement of paramyosin molecules in
the thick filament. The paramyosin molecules assemble into fibers with an
axial repeat of 72.5 nm and staggering of these filaments generates the
characteristic `checkerboard' array of nodes. In negatively stained samples
the nodes are the gaps between molecules where stain is trapped
(Squire, 1981;
Cohen, 1982).
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