Bf. Mcewen et al., A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution, CHROMOSOMA, 107(6-7), 1998, pp. 366-375
Three decades of structural analysis have produced the view that the kineto
chore in vertebrate cells is a disk-shaped structure composed of three dist
inct structural domains. The most prominent of these consists of a conspicu
ous electron opaque outer plate that is separated by a light-staining elect
ron-translucent middle plate from an inner plate associated with the surfac
e of the pericentric heterochromatin. Spindle microtubules terminate in the
outer plate and, in their absence, a conspicuous corona of fine filaments
radiates from the cytoplasmic surface of this plate. Here we report for the
first time the ultrastructure of kinetochores in untreated and Colcemid-tr
eated vertebrate somatic (PtK1) cells prepared for optimal structural prese
rvation using high-pressure freezing and freeze substitution. In serial thi
n sections, and electron tomographic reconstructions, the kinetochore appea
rs as a 50-75 nm thick mat of light-staining fibrous material that is direc
tly connected with the more electron-opaque surface of the centromeric hete
rochromatin. This mat corresponds to the outer plate in conventional prepar
ations, and is surrounded on its cytoplasmic surface by a conspicuous 100-1
50 nm wide zone that excludes ribosomes and other cytoplasmic components. H
igh magnification views of this zone reveal that it contains a loose networ
k of light-staining, thin (<9 nm diameter) fibers that are analogous to the
corona fibers in conventional preparations. Unlike the chromosome arms, wh
ich appear uniformly electron opaque, the chromatin in the primary constric
tion appears mottled. Since the middle plate is not visible in these kineto
chore preparations this feature is likely an artifact produced by extractio
n and coagulation during conventional fixation and/or dehydration procedure
s.