Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.

Abstract

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores ." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed . Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 p,m in diameter. Preabsorption of the antisera was attempted using microtubule protein, purified tubulin, actin, and microtubule-associated proteins . None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region . In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.The centromere or kinetochore region in most animal cells can be seen in the light microscope as a localized constriction appearing on otherwise linear metaphase chromosomes . It is at this region that the sister chromatids, attached along their entire lengths, first separate at the onset of anaphase. The region also functions as the site of spindle fiber (microtubule) attachment. The placement of the centromere on the chromosome may differ from chromosome to chromosome, but there is a constant and recognizable location on homologous chromosomes and a specific pattern within a given species .In the electron microscope, the mitotic centromere is also recognized by its constricted configuration and by the presence THE JOURNAL OF CELL BIOLOGY " VOLUME 91 OCTOBER 1981 95-102 © The Rockefeller University Press -0021-9525/81/10/0095/08 $1 .00 in some species of a distinct, specialized structure called the kinetochore . The ultrastructure of the kinetochore varies in plant and animal species and has been especially well described in mammalian cells (7) . Briefly, it appears to have a trilaminar morphology in cross section, with an electron-dense outer plate, lightly staining middle layer, and a darker inner area immediately adjacent to the underlying centromeric heterochromatin . Spindle microtubules are specifically attached to the outer plate . There is also compelling evidence to suggest that kin...

Characterizing a rat Brca2 knockout model

“…Meiosis in Brca2 À/À rats proceeds normally through leptotene and early zygotene (Figure 3a) with 40 centromeres clearly identified by CREST antisera (Brenner et al, 1981). The correct number of centromeres and chromosome cores indicates that sister chromatid cohesion is not affected in the absence of Brca2.…”

Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells

“…The mitotic apparatus is an appealing context for the use of such spatial domain techniques because it contains discrete structures that can be manipulated in order to address their respective roles in the production of pulling or pushing forces to move chromosomes during mitosis. Accordingly, laser microsurgery has been seminal to elucidate the mechanistic basis of the spindle-assembly checkpoint (2), the role of the centrosome in spindle assembly (3) and the role of kinetochores in chromosome movement (4)(5)(6). We believe that we can significantly broaden this scenario by combining powerful live cell imaging and state-of-the-art laser technology with molecular tools.…”

Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments

“…Additional information concerning the distribution of interphasic centromeres and the extent of their possible redistribution between late G 2 and metaphase is paramount when one tries to interprete such data in terms of an interphase chromosome arrangement. For example, it makes an important difference whether centromeres are clustered in a limited portion of the nucleus either at the nuclear edge (Rabl 1885) or elsewhere (Del Fosse and Church 1981) or distributed largely at random (Brenner et al 1981). Recent studies of the distribution of interphasic centromeres indicate considerable variation in cell types from different plant and mammalian species (Church and Moens 1976;Del Fosse and Church 1981;Moroi et al 1981;Brenner et al 1981).…”

“…For example, it makes an important difference whether centromeres are clustered in a limited portion of the nucleus either at the nuclear edge (Rabl 1885) or elsewhere (Del Fosse and Church 1981) or distributed largely at random (Brenner et al 1981). Recent studies of the distribution of interphasic centromeres indicate considerable variation in cell types from different plant and mammalian species (Church and Moens 1976;Del Fosse and Church 1981;Moroi et al 1981;Brenner et al 1981). Thirdly, and probably most important, the technical procedures routinely used for obtaining well spread metaphases (often including colchicine treatment and hypotonic shock) alter the chromosome arrangement of the intact metaphase plate to an extent which has not been documented by most investigators, although it can be shown that the relationship between chromosome arrangements at interphase and metaphase is critically influenced by the experimental regimen followed (Schmid et al 1981).…”