Cellular behavior and centriole distribution during multipolar mitosis induced by nocodazole action

After recovery from the nocodazole blockade, mitoses in PE cells proceed differently depending on the time of treatment and on the drug concentration. Cells, treated with 0.02 mcg/ml for 3 hours or less, have a recovery period of 1-1.5 hours, however cells, treated with 0.02 mcg/ml for more than 3 hours or with 0.2 mcg/ml at a time, have a recovery period of 3-4 hours. In both the cases anaphase and cytokinesis proceed normally. The 0.6 mcg/ml nocodazole concentration results in the appearance of only multipolar mitoses during recovery. The minimal-time multipolarity induction is 1 hour. Cytokinesis is disturbed in 60% of multipolar mitoses: two of the three daughter cells are fused to form a binucleated cell. A complete disruption of the mitotic apparatus causes one of the diplosomes to dissociate. In the first minutes of recovery, the other diplosome dissociates too. In tripolar telophase centrioles distributed among the spindle poles according to the 2 : 2 : 0 pattern, as a rule. Thus, the deranging of the mitotic spindle is a necessary and sufficient condition for the induction of multipolar mitoses in tissue culture cells. This derangement accompanies the dissociation of diplosomes, but single daughter centrioles do not form a spindle pole.     

Structure of the centriolar complex in the hepatocytes of intact and regenerating mouse live

The structure of the cellular center in polyploid hepatocytes of intact and regenerating liver of adult mice has been studied. It was shown that the structure of the centriolar complex depends on stages of the cellular cycle. No pericentriolar structures (such as satellites, appendages and others) and cytoplasmic microtubules were found in the centriolar complex within G0-period. The satellites and appendages are formed in the half of the centrioles within G1-period. The microtubules can branch off some satellites; the daughter centrioles begin to form within S-period; there are diplosomes in the cells within G2-period, some mother centrioles are surrounded with the fine fibrillar halo. It is concluded that the structure of the centriolar complex within G0-period is distinguished by that within G1-period. The structure of the centriolar complex in polyploid hepatocytes has the same feature of reorganization in certain interphase periods of the cell cycle as in diploid cells of some cultured cells and the thyroid epithelium.     

A centriole-free Drosophila cell line. A high voltage EM study

The problem of the absence of centrioles in cells of the 1182-4 line of Drosophila melanogaster has been reexamined with high voltage electron microscopy. A hypotonic treatment of the cells before fixation allowed a clear recognition of centrioles in 1 micron thick sections. Three different approaches were used to determine the presence of absence of centrioles in a Kc control cell line and in the 1182-4D cell line. 1) 1500 random 0.5 micron thick sections representing the equivalent of 60 whole cells of the 1182-4D line show no centrioles. In contrast, nearly all centrioles of the Kc cells were detected by this examination. 2) In a blind test 10 grids with either Kc or 1182-4D cells were correctly identified by the operator. In Kc cells, 4 to 6 diplosomes were observed by grid square on about 300 cell profiles, while no centrioles were seen in the sections of 1182-4D cells. 3) Complete serial sections 1 micron thick of whole 1182-4D cells were screened for presence or absence of centriole. No centriole was seen in any section. We conclude that these Drosophila 1182-4D cells, which have been maintained in culture for several years, are free of centrioles.     

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole.     

Centrin-2 is required for centriole duplication in mammalian cells

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.     

Ultrastructural disorders of the mitotic apparatus during cytochalasin B action

A correlation between the number of chromosome sets and the number of centrioles (8n--8 centrioles) was observed in polyploid metaphase cells, during cytochalasin B treatment on the cultured Chinese hamster cells. There is no correlation between the number of chromosome sets and the centriole number after stopping the action of the drug in many cells, but a great variation is observed in maintenance of chromosomes and centrioles (up 6 to 25 n and up 4 to 22 centrioles). In multipolar mitosis, either during the drug action or after its stopping, different numbers of chromosomes are directed towards the poles not depending on the number of centrioles in the poles. During the cytochalasin B treatment, either in bipolar or multipolar metaphases, there are destructions in the ultrastructure of the mitotic apparatus: there are no astral microtubules; in the poles there are diplosomes and duplex of centrioles with fibrillar material around both centrioles; kinetochores are of prometaphase type. After stopping the drug action the astral microtubules appear, but no other patterns of normalization in the mitotic apparatus occur. Desynchronization of three cycles (chromosomal, centriolar and centrosomal) is discussed as a factor of abnormal development of the mitotic apparatus and as a factor of stabilization of aneuploidy in the cell culture.     

Centriole modification in human aortic endothelial cells.

The structure of centrioles in endothelial cells of embryonic (22-24 weeks old) and definitive (2, 14-17, and 30-40 years) human aorta in situ and also in aortic endothelial cells dividing in organ and cell cultures (donor age 30-40 years) was studied. It was found that in the endothelial cells from definitive aorta the lengths of mother centrioles vary from 0.5 to 2 microns, whereas the length of daughter centrioles remains constant (0.4-0.5 microns). The distal part of the cylinder of long mother centrioles consists of microtubule doublets. In aorta of donors 30-40 years old in multinucleated cells and in one of 30 single-nucleated cells analyzed, C-shaped long centrioles were seen. These centrioles exhibit a doublet organization along all their length. Mitotic cells in organ and cell culture had a nonequal structure of spindle poles: at one pole, the long mother centriole was seen, while at the other a mother centriole of standard size was found. In such cells of organ culture long centrioles make contact with the remnant of primary cilia until the end of anaphase. In cell culture mitotic cells are also observed containing C-shaped centrioles. In these cells the number of mother centrioles is odd and their number is not equal to the number of daughter centrioles. The possible mechanism for transformation of endothelial centrioles and its role in the control of cell-cycle progression are discussed.     

Centriole as microtubule-organizing centers for marginal bands of molluscan erythrocytes

The erythrocytes of blood clams (arcidae) are flattened, elliptical, and nucleated. They contain elliptical marginal bands (MBs) of microtubules, each physically associated with a pair of centrioles marginal bands (MBs) of microtubles, each physically associated with a pair of centrioles (Cohen, W., and I. Nemhauser, 1980, J. Cell Biol., 86:286-291). The MBs were found to be cold labile in living cells, disappearing within 1-2 h at 0 degrees C. After the cells had been rewarmed for 1-2 h, continuous MBs with associated centrioles were once again present. Time-course studies utilizing phase contrast, antitubulin immunofluorescence, and electron microscopy of cytoskeletons prepared during rewarming revealed structural evidence of centriole participation in MB reassembly. At the earliest stage of reassembly, a continuous MB was not present. Instead, relatively short and straight microtubules focused on a pointed centriolar “pole,” and none were present elsewhere in the cytoskeleton. Thin continuous MBs then formed, still pointed in the centriolar region. Subsequently, the MBs regained ellipticity, with their thickness gradually increasing but not reaching that of controls even after several hours of rewarming. At these later time points, microtubules still radiated from the centrioles and joined the MBs some distance away. In the presence of 0.1 mM colchicines, MB reassembly was arrested at the pointed stage. Electron microscopic observations indicate that pericentriolar material is involved in microtubule nucleation in this system, rather than the centriolar triplets directly. The results suggest a model in which the centrioles and associated material nucleate assembly and growth of microtubules in diverging directions around the cell periphery. Microtubules of opposite polarity would then pass each other at the end of the cell distal to the centrioles, with continued elongation eventually closing the MB ellipse behind the centriole pair.     

Induction of multipolar mitoses in cultured cells: Decay and restructuring of the mitotic apparatus and distribution of centrioles

During recovery after a long (up to 12 h) treatment of pig embryo culture cells (PK) with nocodazole at concentrations of 0.02 g/ml and 0.2 g/ml all c-metaphase cells divide normally into two daughter cells. During recovery after a short (1–4 h) treatment with 0.6 g/ml nocodazole only multipolar mitoses (as a rule tripolar) arise. At the ultrastructural level, the increasing nocodazole concentration leads to progressive disruption of the mitotic spindle. At a nocodazole concentration of 0.2 g/ml kinetochores are not associated with microtubules. At a nocodazole concentration of 0.6 g/ml there are no microtubules around the centrosomes, and in every cell one of the two diplosomes disintegrates. In tripolar telophase centrioles are distributed among the spindle poles generally in a 2:2:0 pattern. Mother and daughter centrioles are always disoriented but not separated. The centriole-free pole contains a cloud of electron-dense material. During tripolar division two of the three daughter cells mainly fuse shortly after telophase forming one binucleate cell. Thus a multipolar mitosis arises as a result of the uncoupling of mother centrioles and spindle microtubules, but not of the duration of the c-mitotic arrest. Centriole-free poles account for the divergence of chromosomes, but mainly they are unable to ensure the normal cytokinesis of daughter cells.by M. Trendelenburg     

Behavior and function of paternally inherited centrioles in brown algal zygotes

In brown algal cells, the centrosome, consisting of a pair of centrioles and the pericentriolar material, is primarily involved in the organization of microtubules (MTs) throughout the cell cycle. In motile cells, the centrioles participate in the formation of flagellar axoneme as flagellar basal bodies, and in somatic cells they play a crucial role in many cellular activities as a part of the centrosome. With respect to the role of the centrosome as a microtubule organizing center (MTOC), brown algal cells resemble animal cells. In most animal fertilization processes, the sperm cell introduces centrioles, the core of the centrosome, into the egg cytoplasm. In this study, the behavior of centrioles from gametogenesis and fertilization to the first cell division of the zygote was examined in the three sexual reproduction patterns occurring in brown algae, i.e., oogamy, anisogamy and isogamy, by electron- and immunofluorescence-microscopy. The pair of centrioles contained in somatic cells was shown to be derived from the male gamete, irrespective of the sexual reproductive pattern. The paternally derived centrioles were duplicated before mitosis and were involved in spindle pole formation. Moreover, MTs from the centrosome play a crucial role in the process of cytokinesis, as the position of centrosomes accompanying daughter nuclei seems to determine the cytokinetic plane. A new approach to clarifying the mode of cytokinesis in brown algae is presented in this study.Chikako Nagasato was the recipient of the Botanical Society of Japan Award for Young Scientist, 2004.     

Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus

Ultrastructural changes in the maturing oocyte of the sea urchin Hemicentrotus pulcherrimus were observed, with special reference to the behavior of centrioles and chromosomes, using oocytes that had spontaneously started the maturation division process in vitro after dissection from ovaries. The proportion of oocytes entering the maturation process differed from batch to batch. In those eggs that accomplished the maturation division, it took ~4.5-5 h from the beginning of germinal vesicle breakdown to the formation of a second polar body. Serial sections revealed that a young oocyte before germinal vesicle breakdown had a pair of centrioles with procentrioles, located between the presumed animal pole and the germinal vesicle and accompanied by amorphous aggregates of moderately dense material and dense granules (granular aggregate). Just before germinal vesicle breakdown, a pair of fully grown centrioles located in the granular aggregate, which is present until this stage and then disappears, had already separated from another pair of centrioles. In meiosis I, each division pole had two centrioles, whereas in meiosis II each had only one. The two centrioles in the secondary oocyte separated into single units and formed the mitotic figure of meiosis II. The first polar body had two centrioles and the second had only one. The two centrioles in the first polar body did not form the mitotic figure nor did they separate at the time of meiosis II. These results indicate that, in sea urchins, duplication of the centrioles does not occur during the two successive meiotic divisions and the egg inherits only one centriole from the primary oocyte, confirming the results previously reported for starfish oocytes.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

GIANT CENTRIOLE FORMATION IN SCIARA

Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop giant centrioles with 60–90 singlet tubules disposed in an oval array. Some 9-membered centrioles still may be seen in second instar spermatogonia. Each of these centrioles is associated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles of second instar spermatogonia consist of 20–50 singlet tubules arranged in an oval, sometimes associated with an even larger secondary centriole. The more recently formed centriole of a pair is distinguishable from its partner by a concentric band of electron-opaque material inside its tubules. If a pair of centrioles at right angles to each other is pictured as a "T" formed by two cylinders, the secondary centriole is always the stem of the T; the primary centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a new daughter centriole. A Golgi area of characteristic morphology is found in association with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole may give rise to a daughter morphologically different from itself. Whether the daughter is a 9-membered or giant centriole depends on the tissue type and stage of development.     

Spermatogenesis and spermiogenesis in Ascaris lumbricoides Var. suum

Reorganization of the prophase I nucleus marks the beginning of the first meiotic division. A pair of centrioles is present at each pole at metaphase I and mitochondria are not observed in the spindle area. A chromosomal pellicle, which resembles a kinetochore plate but has no apparent association with microtubules, surrounds each autosome at metaphase I and II. The sex body lags behind the autosomes at anaphase I and segregates differentially to one daughter cell. Mitochondria and a pair of centrioles are present in the spindle during the second meiotic division. Localized condensation of chromatin and fusion of the condensed chromatin of the secondary spermatocyte telophase nucleus results in a compact spermatid nucleus. Loss of spermatid cytoplasm is effected by the ejection of a cytophore vesicle.     

Centrioles in the cell cycle of L cells

The structure of centrosome in non-synchronous L-cells culture during the cell cycle has been studied. In mitosis, mother and daughter centrioles, which differ in their ultrastructure, are located perpendicularly in the pole of the spindle. Microtubules, meeting in the pole area terminate mainly in electron-dense clottings of fibrillar matter surrounding the diplosoma. In telophase, disjunction of mother and daughter centrioles begins. At the beginning of G1-period, centrioles move off from each other for several micron, and then draw together again without forming diplosome. Pericentriolar satellites form on mother centriole of some cells at this time, they disappear at the beginning of S-period, replication of centrioles begins; daughter centrioles reach the size of mother centrioles in anaphase. During growth and maturation, centrioles in L-cells undergo structural changes similar to those described for SPEV cells (Vorob'ev, Chentsov, 1982). Several types of meeting points for microtubules exist in L-cells during the whole interphase: surface of centrioles per se, pericentriolar satellites, free foci.     

The acentriolar state of the Drosophila cell lines 1182

A Drosophila melanogaster cell line devoid of centrioles has been recently described. In order to achieve an easier characterization of these acentriolar cells, we used the monoclonal antibody Bx 63 of M. Frasch which recognizes the Drosophila centrosome. Although centrosomes are detected at every mitotic pole in Drosophila cells with centrioles, no such structure has been observed in 1182-4 acentriolar cells. The antigenic material is, however, present in these cells. Moreover, we noticed a certain proportion of acentriolar cells in 4 other 1182 lines. The lack of centrioles previously found only in the 1182-4 cells seems therefore not accidental and should be linked to their particular origin.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

Ultrastructure of segmentation mitoses in cleaving newt eggs after blocking of centrospheres by quinoline

In quinoline treated blastomeres of Triturus helveticus Raz. and Pleurodeles waltlii Michah., microtubules disappear and around the centrioles markedly enlarged dense bodies accompanied by striated bodies are accumulated, from prophase beyond metaphase. It is concluded that these bodies in untreated cells play a role in the formation of the spindle microtubules. — During metaphase the chromosome are smooth-surfaced and show a pronounced tendency to stickiness. During ana-telophase they become surrounded by nuclear membranes and form caryomeres. — Quinoline does not interfere with centriole replication, but prevents the separation if diplosomes that have been already formed, if it acts before that separation. — Some centrioles exhibit different degrees of ultrastructural disarrangement.     

Strongylocentrotus drobachiensis oocytes maintain a microtubule organizing center throughout oogenesis: implications for the establishment of egg polarity in sea urchins

Although it has been known for over a century that sea urchin eggs are polarized cells, very little is known about the mechanism responsible for establishing and maintaining polarity. Our previous studies of microtubule organization during sea urchin oogenesis described a cortical microtubule-organizing center (MTOC) present during germinal vesicle (GV) migration in large oocytes. This MTOC was localized within the future animal pole of the mature egg. In this study we have used electron microscopy and immunocytochemistry to characterize the structure of this MTOC and have established that this organelle appears prior to GV migration. We show that the cortical MTOC contains all the components of a centrosome, including a pair of centrioles. Although a centrosome proper was not found in small oocytes, the centriole pair in these cells was always found in association with a striated rootlet, a structural remnant of the flagellar apparatus present in precursor germinal cells (PGCs). The centrioles/striated rootlet complex was asymmetrically localized to the side of the oocyte closest to the gonadal wall. These data are consistent with the previously proposed hypothesis that in echinoderms the polarity of the PGCs in the germinal epithelium influences the final polarity of the mature egg.     

Ultrastructural Studies on the Behavior of Centrioles during Meiosis of Starfish Oocytes

The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis . Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of Pisaster ocraceus and Asterias forbesi . The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte.