The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells . The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early Gl nucleus, where they were found throughout interphase . Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis . Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in Gt or early in S . During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length . To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99 % . Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected . However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.

Centriole formation was studied after inhibition of protein synthesis for various portions of the cell cycle. Synchronous populations of mitotic L929 (mouse) cells were plated into petri dishes and the course of procentriole formation was monitored by electron microscope analysis. The frequency with which procentrioles were seen in association with mature centrioles normally increased steadily in the interval from 4 to 12 h after mitosis. The formation of procentrioles was abruptly inhibited by the addition of cycloheximide at any time from mitosis until 12 h postmitosis (S phase). This suggested that the formation of procentrioles was dependent upon protein synthesis immediately before their appearance. Prophase-associated elongation of procentrioles appeared to occur normally in cells treated with cycloheximide for up to 4 h before prophase, though the mitotic index in treated cultures decreased somewhat. Thus, protein synthesis did not appear to be essential for procentriolar elongation to the mature length.The synthesis of centrioles is under tight control by the cell. Generally, a cell makes exactly two centrioles in the course of a cell cycle. Greater numbers of centrioles may be generated if needed as basal bodies when the cell is preparing to generate cilia (5, 10, 25). The time of centriole production has been worked out in proliferating cultured fibroblastic cells (15, 16; see reference 3 for review). It is found that each daughter cell of a division receives a pair of orthogonally arranged, full-length (about 0.55 /zm long) centrioles. The two centrioles separate from each other in G1 and, near the onset of S phase, one daughter procentriole appears adjacent and perpendicular to each mature centriole. The procentrioles are about half the length of a mature centriole and remain that length until prophase, when they elongate to the mature length (15,17). Thus, the pair of centrioles at the pole of the metaphase spindle again consists of two full-length centrioles. The mechanism by which the cell maintains such tight control on the time of centriole duplication and the quantity and size of procentrioles is not known.In an earlier study (15), we used an inhibitor of DNA synthesis to dissociate the event of nuclear DNA synthesis from procentriole production. It appeared that procentriole formation occurred independently of whether or not DNA synthesis was allowed to proceed. On the other hand, the process of elongation of procentrioles to their mature length appeared to be strictly correlated with the onset of prophase events. In this paper, we present the results of experiments which test the requirements for protein synthesis in procentriole

Chinese hamster cell strains in the early passages in culture display wide variation in number of nucleolus-like bodies per cell, though such strains are characteristically euploid. A variety of criteria indicate that the nucleolus-like bodies are true nucleoli. Their Azure B-and fast green-staining properties indicate the presence of RNA and protein; they have typical nucleolar fine structure, including both fibrous and granular components; radioautography reveals that their patterns of uptake of uridine-3H into RNA are similar to those reported for nucleoli of other cell types; actinomycin D, at a level which selectively inhibits ribosomal RNA synthesis, greatly reduces their RNA synthesis and also causes segregation of fibrous and granular nucleolar components. Colchicine was used to experimentally fragment the nuclei of these cells into a number of separate karyomeres, each presumably containing some, or only one, of the chromosomes of the complement. Almost all the karyomeres contain nucleolus-like bodies which, by the same criteria applied to the multiple nucleolus-like bodies of uninuclear cells, appear to be true nucleoli. The nucleoli of individual karyomeres of the same cell often differ from each other in fine structure while the multiple nucleoli of a uninuclear cell generally resemble each other. The evidence presented in this study indicates that Chinese hamster cells contain many nucleolus-producing sites scattered through the genome.Diploid, nontransformed Chinese hamster cells in tissue culture were found to possess widely varying numbers of nuclear bodies which appeared cytologically to be nucleoli. This finding seemed inconsistent with numerous studies demonstrating that somatic cells are normally characterized by a set number of nucleoli which consistently arise in association with specific chromosomal loci. The number of nucleoli produced is found to be one per haploid chromosome complement, i.e., two per diploid cell, in a great many organisms, for instance, Hyacinthus (de Mol, 1926(de Mol, , 1928,

ABSTRA_CTThis study is concerned with the fate of the nucleolar contents, particuIartv nucleolar RNA, during m~tosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, XB6 (human) cells, or L929 (mouse) cells were allowed to proceed into mterphase in the presence or absence (control) of 0.04-0 08 /~g/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (nbosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04-0 08 #g/ml actinomycm D for 3 hr before harvesting of mitotic ceils, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such ceils were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis

No abstract

Nucleoli of cultured Chinese hamster or mouse cells in early passages had a loosely reticular substructure . Within the reticulum small, irregularly shaped, light fibrillar zones occurred which were contiguous with denser fibrillar zones . These denser zones appeared to be connected in some places to the particulate material which composed the mass of the nucleolus . Generally, electron-transparent spaces separated the particulate zones from the fibrillar areas . Treatment with toyocamycin, an agent which is reported to cause a blockage in the processing of ribosomal RNA, greatly inhibited the accumulation of newly synthesized RNA in the cytoplasm, as monitored by radioautography . Toyocamycin treatment caused the gradual disappearance of the granules from the particulate region of the nucleoli, and resulted ultimately in the nucleoli appearing homogeneously fibrillar. Actinomycin D treatment, which inhibited virtually all RNA synthesis, caused a segregation, and finally a disaggregation, of nucleolar components .

When washed cells from human semen samples were plated out, epithelial cultures were obtained. The human ejaculates used as starting material contained, in addition to spermatazoa, 10(3) to 10(7) cells of other types, including granulocytes, macrophages, lymphocytes, spermatocytes and epithelial cells. Although no fractionation of cell types was attempted, semen samples yielded epithelial cultures uncontaminated by fibroblasts. The cultured cells appeared characteristically epithelial with a polygonal shape, interdigitating cell membranes, and desmosomes. ABH blood-group antigenic determinants of the donor were expressed with variable frequency as a surface antigen on these cells. About half the trials gave some cell attachment. Most cultures remained as small, tight colonies, but a few reached confluency in about 5 weeks and could be subcultured successfully. Cell proliferation, as monitored by (3H) thymidine incorporation into nuclear macromolecules, ceased in less than 2 months.

Nucleoli of cultured cells of the established lines KB and L were found to possess a distinctive fine structural organization. The major portion of the nucleolar volume was composed of compact, particulate material. Spheroidal fibrillar zones about 0.4 µ in diameter occurred within the particulate mass. These fibrillar zones had a central light area and a denser rim. Toyocamycin treatment, which sharply inhibited the appearance of newly synthesized RNA in the cytoplasm, caused the gradual disappearance of the fibrillar material from nucleoli. Actinomycin D treatment, which inhibited virtually all RNA synthesis, caused varying types of segregation of nucleolar components. The morphology of nucleoli of KB and L cells and the reorganization of these nucleoli in response to drugs appear to be different from those of nucleoli of freshly initiated Chinese hamster and mouse cell lines.