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...

New applications of laser microbeam irradiation to cell and developmental biology include a new instrument with a tunable wavelength (217- to 800-nanometer) laser microbeam and a wide range of energies and exposure durations (down to 25 × 10 -12 second). Laser microbeams can be used for microirradiation of selected nucleolar genetic regions and for laser microdissection of mitotic and cytoplasmic organelles. They are also used to disrupt the developing neurosensory appendages of the cricket and the imaginal discs of Drosophila .

The number, distribution, and nucleating capacity of microtubule-organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells . Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material . MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm . The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCS was assayed by incubating tubulin-depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 6S brain tubulin and microtubule assembly buffer . Initiation and assembly of 6S tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific . Our results are consistent with the notion that the specification of microtubule length, number, and spacial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits .

Antibodies from the serum of patients with the autoimmune disease scleroderma CREST were used to investigate the association and distribution of kinetochores in mouse cells during meiosis and spermiogenesis. The pattern of indirect immunofluorescent staining in pachytene nuclei indicated that each autosomal bivalent contains one fluorescent spot. Throughout pachytene, the kinetochores were arranged non-randomly into several clusters and distributed around the periphery of the nucleus. In subsequent stages of meiotic prophase I, distribution was random and the number of fluorescent spots increased from 21 to 40 corresponding to the diploid chromosome number and the number of halfbivalents oriented to the spindle poles at the metaphase I. Twenty pairs of kinetochores were observed at metaphase II. During spermiogenesis, the number of kinetochores correlated with the haploid chromosome number in early spermatids but tandem association of centromeres and clustering into a conspicuous chromocenter corresponded to a significant reduction in the number of fluorescent foci in mid-spermatid nuclei. The number of stained sites per nucleus continued to decrease during sperm maturation and total absence of staining was apparent in mature spermatozoa. Immunoblotting of proteins extracted from mature sperm however, indicated that a kinetochore antigen of Mr 80,000 was still present. Therefore, the absence of kinetochore staining in mature spermatozoa is probably due to the blockage of epitopes during chromatin condensation.

An argon ion laser microbeam (488 and 514 rim) was used to selectively irradiate one of the to centriolar regions of rat kangaroo Potorous tridactylis (PtK2) prophase cells in vitro. The cells were sensitized to the laser radiation by treatment with acridine orange (0.1-0.2 /.tg/ml). Ultrastructural examination of the irradiated centriolar regions demonstrated that the primary site of damage was the pericentriolar material. This result suggests that nucleic acid is present in the pericentriolar material. Behavioral and ultrastructural analysis demonstrated that cells with one damaged pericentriolar zone could undergo (a) nuclear membrane breakdown, (b) chromosome condensation, (c) metaphase plate formation, and (d) cytokinesis. However, the chromosomes neither separated nor exhibited any anaphase movements. Detailed ultrastructural analysis revealed the presence of kinetochore microtubules on both sides of the chromosome mass and a lack of microtubules in the cytokinesis constriction. These results indicate that the pericentriolar material is important in spindle organization and essential for the formation of the interpolar microtubules.