Kinetochore Structure
Each replicated chromosome possesses two discrete "sister" kinetochores that are positioned on the opposite sides of its primary constriction. During mitosis, sister kinetochores firmly attach their associated chromosome to the spindle by capturing the plus ends of dynamically unstable microtubules (Mts) growing from the poles. Individual kinetochores then use these kinetochore fiber (K-Fiber) Mts as a scaffold for producing much of the force for chromosome poleward motion, and during this motion hold onto the ends of associated Mts as they grow and shrink by Mt subunit addition/dissociation within their confines. In addition to roles in chromosome attachment and force production, kinetochores also control the timing of anaphase onset by producing an inhibitor of this event until the kinetochore is properly attached to the spindle.
The mechanisms by which the kinetochore accomplishes its various functions are not apparent from its structure. In sections from conventionally fixed and stained vertebrate somatic cells unattached kinetochores are seen to consist of a circular 35-40 nm-thick electron opaque plate-like structure that consists of a dense meshwork of 10-20 nm thick fibers. This plate is separated from the underlying centromeric heterochromatin by a 15-30 nm thick electron-lucent zone relatively devoid of structure. A conspicuous fine fibrillar "corona" material radiates 100 nm or more from the cytoplasmic surface of this plate, which can vary significantly in diameter between the chromosomes of a genome . As this kinetochore plate attaches to Mt ends the corona becomes less distinct , its diameter decreases significantly.
A number of models have been proposed to explain kinetochore function, especially the way this organelle remains attached to growing and shortening Mts as it moves towards and away from its pole, while producing most of the force for chromosome poleward motion . Many of these schemes ascribe different functional roles to each of the three structural domains that form the kinetochore. However, our concept of kinetochore structure is based entirely on fixation procedures developed more than 30 years ago that are known to extract the cytoplasmic components and induce subtle and even not so subtle structural changes. In this study we document our initial observations on the structure of the vertebrate kinetochore in sections cut from cells fixed by high pressure freezing followed by freeze substitution and embedding. This procedure relies on using a high pressure freezing apparatus to prevent ice crystal formation during freezing (i.e., vitreous freezing). Vitreous ice is then replaced with fixative dissolved in a dehydrant at -90o C,(i.e., freeze-substitution). This approach minimizes the displacement, extraction, and coagulation of molecules that occur as a front of buffered chemical fixative sweeps through a cell, and it also minimizes the structural distortions produced in the specimen during dehydration due to changes in surface tension.
Results of our study reveal that the electron translucent kinetochore "middle" layer is greatly exaggerated in conventionally prepared specimens. The data further reveal that the kinetochore consists of fine fibers that radiate directly from the surface of the chromosome and connect with tangential fibers to form an extended mat that becomes condensed into a prominent outer plate in response to conventional fixation and dehydration protocols
Bruce F. McEwen, C-E. Hsieh, A.
L. Mattheyses, and C. L. Rieder.
A new look at kinetochore structure in vertebrate somatic cells
using high pressure freezing and freeze substitution.
Chromosoma 107:366-375 (1998)
Co-winner of the Chromosoma prize 1999
Abstract
Three decades of structural analyses 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 electron translucent "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 (PtK1) 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 (<9nm 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 electron-translucent in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.
Figure 1. Comparison between high-pressure freezing/freeze substitution (HPF/FS) and conventional specimen preparations for colcemid treated PtK1 cells. A. Kinetochore prepared via conventional protocol. The outer plate (op) is a heavily stained, compact, structure that is separated from the underlying heterochromatin by a translucent middle layer (ml). A prominent fibrous corona radiates from the outer plate’s distal surface. Note the general extracted appearance of the surrounding cytoplasm, and the uniform staining of the centromeric chromatin (white arrows). B. Sister kinetochores prepared via HPF/FS. In the top kinetochore, the fibrous mat structure (fm) is lightly stained and much more open than the outer plate in A. The corona appears as a cytoplasmic exclusion zone (black arrows) lacking in discernable substructure. In contrast to conventional preparations, the heterochromatin has a mottled appearance (white arrows) and the surrounding cytoplasm is smooth, uniform, and unextracted. The lower kinetochore is sectioned at an oblique angle. As a result the mat structure is not evident, and was not found in the neighboring serial sections. However, the exclusion zone is visible (arrows). Bar = 250 nm.
Figure 2. Low magnification view of a colcemid-treated PtK1 cell prepared via HPF/FS. Kinetochores are most readily recognized by the adjoining "clear" zones that exclude ribosomes and other cytoplasmic particles. Longitudinally sectioned kinetochores (K1, K2) also show faintly staining mats (black arrows) that are not evident in obliquely sectioned kinetochores (K3-K5). The mottled appearance of centromeric heterochromatin (white arrows) is a general feature of chromosomes prepared via HPF/FS. Bar = 500 nm.
Figure 3. Higher magnification view of a colcemid-treated HPF/FS kinetochore. This chromosome is oriented with a slight rotation into the section plane. The mat structure on the right-hand kinetochore appears to be constructed from fine fibers radiating out from the underlying chromatin (white arrows) and crossing fibers lying parallel to the chromatin surface (black arrows). The mat is not visible in the left-hand kinetochore in this section, but the kinetochore can be detected by the prominent exclusion zone with ribosomes piled up along the boundary (large black arrows). Bar = 200 nm.
Figure 4. The exclusion zone in an obliquely cut section. A. A roughly axial view of the exclusion zone (boundaries marked by large arrows), detected primarily by exclusion of ribosomes and other cytoplasmic material. A fine flocculent material, possibly of fibrous nature, is seen in this area. Presumably this material consists of corona fibers and/or an enface view of the mat structure B. Serial section to A, showing the obliquely sectioned kinetochore. Both the mat structure (small arrows) and exclusion zone (large arrows) can be discerned. Bar = 250 nm
Figure 5. Tomographic reconstructions of colcemid-treated, HPF/FS kinetochores. A. Low magnification view of sister kinetochore used for tomographic reconstruction (box indicates the approximate area of the reconstruction shown in B). As in Figure 3, the chromosome is oriented with a slight rotation into the section plane. Bar = 500 nm. B. A 9 nm thick slice (average of three successive 2.9 nm thick tomograms) from the 3D reconstruction of the boxed area in A. Fibrous elements are evident radiating from the heterochromatin and from the surface of the mat (small white arrows) but low contrast and inherent resolution limits preclude exact measurements of diameters and lengths. In this view the mat structure appears as a long, thin, fiber-like element running parallel to the chromatin surface (black arrows). A striking difference in texture between euchromatin and centromeric heterochromatin is evident along the border of the two domains (large white arrows). Bar = 200 nm. C. Low magnification view of the kinetochore used for the tomographic reconstruction illustrated in D and E (box delineates the reconstructed area). Bar = 500 nm. D. A 9 nm thick slice (like B) from the 3D reconstruction of the boxed area in D. The mat shows a block-like arrangement (black arrows) and some unit blocks having a tripartite organization (white arrows). Bar = 200 nm. E. Tilted view of the mat-structure. The mat has been isolated from the remainder of the 3D reconstruction and is displayed as a semitransparent subvolume using masking tools and volume rendering software (see McEwen et al., 1993; McEwen and Marko, 1998). The block-like arrangement of putative unit structures is particularly evident in this view (arrows). Bar = 50 nm
Figure 6. HPF/FS vs conventional specimen preparations for untreated PtK1 cells. A. Adjacent kinetochores from a conventionally prepared metaphase cell. Note the prominent outer plate (op) structure that stains as heavily as chromatin, and is separated from the underlying inner plate (ip) by a well defined, translucent, middle layer (ml). Microtubule (Mt) plus ends can not be clearly traced within the highly condensed outer plate. B, C. Two different kinetochores from an anaphase cell prepared via HPF/FS. Note the absence of a prominent outer plate. Instead there is a lightly staining mat structure that sits above the chromatin without an intervening translucent layer. Mt plus ends can be traced (small black arrows) and they appear to flay apart and be continuous with the fibrous mat (in agreement with Mastronarde et al., 1997). The cytoplasmic exclusion zone can still be detected in some areas where there are no Mts (large black arrows). The mottled appearance of the centromeric heterochromatin is also evident (white arrows). Bar = 200nm.