A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

Citation
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
Citations number
48
Categorie Soggetti
Molecular Biology & Genetics
Journal title
CHROMOSOMA
ISSN journal
00095915 → ACNP
Volume
107
Issue
6-7
Year of publication
1998
Pages
366 - 375
Database
ISI
SICI code
0009-5915(199812)107:6-7<366:ANLAKS>2.0.ZU;2-C
Abstract
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.