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

Abstract

Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electrontranslucent middle plate from an inner plate associated with the surface 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-treated vertebrate somatic (PtK 1 ) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint control

“…Hec1 recruits Zwint-1 and ZW10 to kinetochores for chromosome segregation It is known that the kinetochores comprise several layers of structures that are likely assembled in a hierarchical order by multiple copies of distinct protein subcomplexes (Brinkley and Stubblefield, 1966;Zinkowski et al, 1991;McEwen et al, 1998;Kniola et al, 2001;De Wulf et al, 2003). Both Hec1 and Zwint-1 are the outer kinetochore components, while ZW10 is present at the fibrous corona (Kitagawa and Hieter, 2001;Chan et al, 2005).…”

Nucleolin functions in nucleolus formation and chromosome congression

“…Electron microscopy studies have revealed that the plus ends of spindle microtubules terminate in the outer kinetochore of vertebrate cells (McEwen et al, 1998;VandenBeldt et al, 2006), suggesting that nucleolin covering the outermost layer of kinetochores could affect the kinetochore-microtubule attachments. We found that cells depleted of nucleolin were delayed at prometaphase with misaligned or non-aligned chromosomes and defects in chromosome biorientation.…”

“…Activation of Aurora-B during mitosis can correct chromosome attachment errors by selective disassembly of kinetochoremicrotubule fibers, thereby activating the spindle checkpoint until all the chromosomes make proper bipolar attachments to the mitotic spindle. It is therefore probable that nucleolin depletion does not activate the checkpoint directly but rather causes an increase in syntelic attachments, which are changed into unattached kinetochores by Aurora-B.Electron microscopy studies have revealed that the plus ends of spindle microtubules terminate in the outer kinetochore of vertebrate cells (McEwen et al, 1998;VandenBeldt et al, 2006), suggesting that nucleolin covering the outermost layer of kinetochores could affect the kinetochore-microtubule attachments. We found that cells depleted of nucleolin were delayed at prometaphase with misaligned or non-aligned chromosomes and defects in chromosome biorientation.…”

CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells

“…The corona on unbound kinetochores was clearly visible and similar to control unbound kinetochores (Figure 2e). Because detergent extraction and conventional fixation mask fine structure alterations (McEwen et al, 1998), we chose not to perform a more detailed analysis at this time. The injected anti-CENP-E antibodies were detected with Cy-5-conjugated anti-rabbit antibodies (c and f).…”