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...

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 .

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes .Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg" buffer . This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phosphocellulose-purified tubulin dimers at 37°C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores . Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed .KEY WORDS Centrosomes " kinetochores microtubule nucleation " tubulin antibody electron microscopy During mitosis, spindle microtubule assembly occurs at specific nucleating sites such as the kinetochore on the chromosomes and the spindle poles (for reviews, see references 1, 15, 17) . Such regions, known widely as microtubule organizing centers (MTOCs) (20), have been described extensively but relatively little is known of their molecular composition or the way in which they initiate the assembly of tubulin into microtubules. Several cytochemical studies have indicated that kinetochores, centrioles (basal bodies), and related MTOCs contain RNA which may be involved in the nucleation of microtubule assembly (2,13, 19,30 could be detected by immunoperoxidase staining with tubulin antibodies. Moreover, it was proposed that tubulin was an intrinsic protein of the MTOC and was essential for the initiation of microtubule polymerization at these sites. To further evaluate the existence of tubulin in MTOCs and to test the notion that intrinsic tubulin of kinetochores is essential for microtubule polymerization, we investigated the assembly of purified 6S bovine brain tubulin at kinetochores and centrioles of lysed mitotic cells before and after exposure to tubulin antibodies . MATERIALS AND METHODSTubulin Purification and Characterization J. CELL BIOLOGY © The Rockefeller University Press -0021-9525/79/08/0585/07 $1 .00Microtubule protein was prepared from bovine brain by three cycles of polymerization-depolymerization according to the method of Borisy et al. (5) . The microtubule pellet obtained was resuspended in cold buffer A

Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK1) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 A thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.

A lysed cell system was used to study the organelle structure and nucleation of exogenous tubulin at kinetochores and centrosomes in mitotic PtK2 cells. We have used this lysed cell system in conjunction with nuclease digestion experiments to determine which specific nucleic acids (DNA or RNA) are involved in either the structure and/or microtubule-initiating capacity of kinetochores and centrosomes. The results indicate that DNase I specifically decondenses the kinetochore plate structure, with the eventual loss in the ability of the chromosomes to nucleate microtubule assembly. DNase I had no effect on either the structure or nucleating capacity of centrosomes. Both RNase T1 and RNase A specifically attacked the amorphous pericentriolar material of the centrosomes, with a concomitant loss in the ability of this material to nucleate microtubule formation. Neither RNase appeared to affect the structure or nucleating capacity of the kinetochore. Therefore, the two types of nucleases appear to exert preferential effects on the different types of microtubule initiation sites in mitotic mammalian cells. The results suggest that DNA is a major component of the kinetochore, while RNA is a major component of the amorphous pericentriolar material. These findings support the concept that microtubule initiation sites in mitotic cells contain nucleic acids which are essential for the structural and functional integrity of the sites.