Light and electron microscopy of Rat kangaroo cells in mitosis

The role of the kinetochore in chromosome movement was studied by 532-nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 p,m and remain parallel to each other, not undergoing any poleward separation . Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it . Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of -3 ,um/ min . If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole . The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid . Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it .It is now generally accepted that mitotic spindle formation and chromosome movement involve several structures, including microtubules, centrioles, pericentriolar regions, and the centromere regions of the chromosomes . There have been several investigations into the ultrastructure and chemical composition of the kinetochore, or attachment site of microtubules to the centromeric region (14,(30)(31)(32)35) . Electron microscope studies have revealed a relatively uniform morphology of the kinetochore region in many different eukaryotes (including PtK 2 cells [34]) consisting of a trilaminar structure 0 .3-0 .6 ttm in diameter.Although the kinetochore has been demonstrated unequivocally to be a microtubule-organizing center in vitro (15,22,36,38), very little biochemical information is available on it . However, there are indications that ribonucleoprotein is a component of the kinetochore (9, 32) and it appears to be associated with the inner plate of the trilaminar kinetochore in PtK, cells .One of the earliest studies on the function of the centromere pertains to the mitotic behavior of x-ray-induced chromosome fragments lacking centromeres (11,12). Although these fragments did not join the metaphase plate, there was some sepa-THE JOURNAL OF CELL BIOLOGY " VOLUME 88 MARCH 1981 543-553 ©The Rockefeller University Press " 0021-9525/81/03/0543/11 $1 .00 ration of the chromatids at anaphase. However, they lagged behind the...

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“…The velocity is approximately the same in both directions . (34,35) . Although this outer layer appears intact in Fig.…”

Section: Discussionmentioning

confidence: 99%

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells.

McNeill¹,

Berns²

1981

The Journal of Cell Biology

131

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“…PtK 1 cells that were to be fixed by conventional fixation protocols were grown on coverslips, with or without Colcemid, and then fixed as described previously (e.g., Roos 1973;McEwen et al 1997). Briefly, the cultures were fixed for 30 min with 2.5% glutaraldehyde in phosphate buffer, pH 7.2, postfixed for 60 min with 2% OsO 4 in deionized distilled water, washed, treated for 1 min with 0.15% tannic acid, and stained en bloc for 1-3 h with 1% uranyl acetate in dH 2 0.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1998

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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.

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“…Since the pathway to bipolar orientation during astral mitosis com- monly involves an initial transient mono-orientation intermediate, a mono-orientation cannot be considered aberrant unless it persists until anaphase. Over the years, scattered EM observations, presented parenthetically in the context of larger studies, have shown that both sister kinetochores of a mitotic prometaphase chromosome can be oriented to the same pole [e.g., Bajer and Molk-Bajer, 1969;Roos, 1973;Rieder and Borisy, 19811 (Fig. 2).…”

Section: Chromosome Mal-orientation During Mitosismentioning

confidence: 99%