Centriolar complex in somatic cell hybrids (mouse X Chinese hamster)

The ultrastructure of centriolar complex in interphase cells and in for a long time dividing somatic hybrid cells (mouse X Chinese hamster) has been investigated. It was found that in the majority of hybrid cells (about 80%) the centriolar complex consists of a higher quantity of centrioles than in the parent cells; the fine structure of the centriolar complex has features characteristic of the centrioles of both the parents; the numerous centrioles have a capacity of organizing individual poles of the spindle of division to constitute the ground for multipolar mitosis in the hybrid cells. Besides that, hybrid cells were found with the centriole number corresponding to that in diploid cells. According to our preliminary data, the ultrastructure of these hybrid centrioles is like that in murine cells.     

INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

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 G₁ 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 G₁ 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.     

The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells

Dividing cells in monolayers of the rat-kangaroo (Potorous tridactylis) cell line Pt-K₁ have large spindles and are flat, thus making possible studies of interactions between the achromatic and chromatic parts of the mitotic apparatus during the cell cycle. At prophase, asters and centrioles seem to exert pressure on the nuclear membrane leading to its rupture and penetrance of the centrioles. Apparently, the long axis of the spindle is shorter than the nuclear diameter. What appears as persistent, large portions of the nuclear membrane were observed in some metaphase and anaphase cells. Such a condition might also indicate an arrested mitosis. The midbody, which was often bipartite, was found to be of a ribonucleoprotein nature. — Three-group metaphases were of common occurrence and might represent early stages of chromosome orientation preceding the final alignment of the chromosomes on the equatorial plate. They could also be an expression of an anomalous condition as a result of mitotic arrest during prometaphase owing to spindle inactivation or breakage, errors in centromere-spindle attachments, interference with chromosome movement, or a duplicated centriolar constitution. Most of these aberrations could be attributed to the flatness of dividing cells, which might also bring about the failure of centriole separation and spindle organization in prometaphase stages, as well as multipolar mitosis.De novo organization of half spindles might take place in cells with ruptured spindles. Anaphase cells showing signs of a previous three-group orientation were rare. — Multipolar mitoses were prevalent mainly in cells with high chromosome numbers. They were often star-shaped with the chromosomes oriented between opposite and adjacent poles, and rarely as end-to-end associations of spindles. Apparently, one or more centrioles might share a common polar region. Multipolar configurations have either a mono- or multinuclear origin. Nuclei usually enter division synchronously in binucleate cells and the spindles become organized between centrioles associated with individual or different nuclei.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

Laser microbeam irradiation of rat kangaroo cells (PTK2) following selective sensitization with bromodeoxyuridine and ethidium bromide

Ethidium bromide (l0 μg/ml) and bromodeoxyuridine (25 μg/ml) were used to sensitize selective cell organelles to visible wavelengths of an argon ion Her (488 and 514 nanometers). Ethidium bromide was shown to be salabtlve In sensitizing nucleoli, chromosomes, and the centriolar region of PTK₂ cells to the laser microbeam. Similarly, BrDU sensitized chromosomes to the microbeam irradiation. The lesions produced on the chromosomes when either agent was used appeared as a phase paling of the irradiated segment. Nucleolar lesions also appeared as a phase paling, and the centriolar region alteration appeared either as a phase paling or a phase darkening.     

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 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     

The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro

Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

Centriole duplication in PE (SPEV) cells starts before the beginning of the DNA replication

Centrosome includes two centrioles and is a structural basis of mitotic spindle pole. Duplication of this organelle and doubling of chromosomes quantity during DNA replication are two principal events of cell cycle in the course of preparation for cell division. In this work, cells of pig kidney embryonic cell line PE (SPEV) were individually monitored after mitosis and procentriole appearance was detected by electron microscopy as soon as 5–6 h after mitosis. This period was 1–2 h shorter than minimal duration of G₁-phase in PE cell line. Ultrastructural analysis of centrosomes in the cells with known “cell cycle age” in combination with autoradiography study of the same cells using ³H-thimidine directly confirmed that duplication of centrioles started earlier than cells entered in S-phase of cell cycle, i.e., preceded the DNA replication.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

The Fine Structure of the Centriolar Apparatus and Associated Structures in the Flagellates Deltotrichonympha and Koruga. II. Division

SYNOPSIS. At division of Deltotrichonympha operculata and Koruga bonita from the Australian termite, Mastotermes darwiniensis , the 2 centriolar bodies separate, each becoming a mitotic center. Spindle microtubules develop from the lower end of each centriolar body and radiate towards the elongating nucleus. A new rostrum is formed in association with each centriolar body. Thus, centriolar bodies which lack the structure of typical centrioles can nevertheless function as division centers during mitosis.     

Cytoplasmic activation of human nuclear genes in stable heterocaryons

We have induced the stable expression of muscle-specific genes in human nonmuscle cells. Normal diploid human amniocytes were fused with differentiated mouse muscle cells by using polyethylene glycol. The fusion product, a stable heterocaryon in which the parental cell nuclei remained distinct, did not undergo division and retained a full complement of chromosomes. This is in contrast with typical interspecific hybrids (syncaryons), in which the parental nuclei are combined and chromosomes are progressively lost during cell division. The human muscle proteins, myosin light chains 1 and 2, MB and MM creatine kinase and a functional mouse-human hybrid MM enzyme molecule were detected in the heterocaryons. Synthesis of these proteins was evident 24 hr after fusion and increased in a time-dependent manner thereafter. Our results indicate that differentiated mouse muscle nuclei can activate human muscle genes in the nuclei of a cell type in which they are not normally expressed, and that this activation occurs via the cytoplasm. The activators are still present in cells which have already initiated differentiation, are recognized by nuclei of another species, and do not diffuse between unfused cells. The reprogrammed amniocyte nuclei of stable heterocaryons provide a unique system in which to study the mechanisms regulating gene expression during cell specialization.     

Events associated with the initiation of mitosis in fused multinucleate HeLa cells

Large multinucleate (LMN) HeLa cells with more than 10–50 nuclei were produced by random fusion with polyethylene glycol. The number of nuclei in a particular stage of the cell cycle at the time of fusion was proportionate to the duration of the phase relative to the total cell cycle. The fused cells did not gain generation time. Interaction of various nuclei in these cells has been observed. The nuclei initially belonging to the G1-or S-phase required a much longer time to complete DNA synthesis than in mononucleate cells. Some of the cells reached mitosis 15 h after fusion, whereas others required 24 h. The cells dividing early, contained a larger number of initially early G1-phase nuclei than those cells dividing late. The former very often showed prematurely condensed chromosome (PCC) groups. In cells with a large number of advanced nuclei the few less advanced nuclei could enter mitosis prematurely. On the other hand, the cells having a large number of nuclei belonging initially to late S-or G2-phase took longer to reach mitosis. These nuclei have been taken out of the normal sequence and therefore failed to synthesize the mitotic factors and depended on others to supply them. Therefore the cells as a whole required a longer period to enter mitosis. Although the nuclei became synchronized at metaphase, the cells revealed a gradation in prophase progression in the different nuclei. At the ultrastructural level the effect of advanced nuclei on the less advanced ones was evident with respect to chromosome condensation and nuclear envelope breakdown. Less advanced nuclei trapped among advanced nuclei showed PCC and nuclear envelope breakdown prematurely, whereas mitotic nuclei near interphase or early prophase nuclei retained their nuclear envelopes for a much longer time. PCC is closely related to premature breakdown of the nuclear envelope. Our observations clearly indicate that chromosome condensation and nuclear envelope breakdown are two distinct events. Kinetochores with attached microtubules could be observed on prematurely condensed chromosomes. Kinetochores of fully condensed chromosomes often failed to become connected to spindle elements. This indicates that the formation of a functional spindle is distinct from the other events and may depend on different factors.     

Control of daughter centriole formation by the pericentriolar material

Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.     

MITOSIS AND CLAMP FORMATION IN THE FUNGUS PORIA MONTICOLA

Nuclear division in P. monticola is in general similar to mitosis in higher organisms. Synchronous division of the nuclei in the dikaryon progresses with clamp development. Mitosis begins with the movement of the centriolar plaques into and under the forming clamp. The pull of the centriolar plaque on the attached nucleolus forms a long strand of nucleolar material. Chromosomes now appear as dense granules at the end of the nucleus proximal to the clamp. At this time the nucleolus moves adjacent to the centriolar plaque and contracted chromosomes. The nuclear membrane at least partially disintegrates, and the nucleolus is released into the cytoplasm where it may persist through telophase. A faintly staining spindle is often observed, and it produces a “double bridge” effect in separating chromatin. Somatic chromosomes are attached together forming strings that appear double and at least partially separated before metaphase.     

Use of enlarged cells and nuclei for studying mitosis inNeurospora

Summary Mitotic division stages studied by light microscopy in differentNeurospora crassa cell types clearly resemble prophase, metaphase, anaphase, and telophase stages of higher eukaryotes. 1. When conidia are cultured in liquid medium containing 3.22 M ethylene glycol, they grow without cell division, forming giant spheres with multiple nuclei. In a few giant cells, nuclear numbers remain small (1 to 3) but the nuclei become very large. Seven large chromosomes are seen in some nuclei suggesting polyteny, 14 or more chromosomes are seen in other, very large nuclei, indicating polyploidy. Cell volume and nuclear volume are positively correlated in giant cells. Nuclear divisions are not synchronous within individual multinucleate giant cells. 2. Nuclear division stages were also observed in crosses heterozygous for the dominant mutant banana where haploid prefusion nuclei in late-forming croziers revert to mitosis. Swollen ascogenous hyphae become highly multinucleate after several rounds of mitosis. Mitosis is completely synchronous in nuclei of the same crozier cyst, providing replicate information for unambiguous identification of division stage. 3. Observations are also reported of mitosis in a cell-wall deficient slime strain. Previous observations on mitosis in large nuclei of the ascus are summarized for comparison. The nucleolus persists throughout mitosis in the giant cells, multinucleate reverted croziers, and in the cell-wall deficient slime strain. It is expelled from the dividing nuclei in the ascus. Spindles and spindle pole bodies, which are normally conspicuous in asci, are also seen in normal and reverted croziers, but they have not been clearly identified in the ethylene glycol-induced giant cells.     

THE CENTRIOLE CYCLE IN SYNCHRONIZED HELA CELLS

Progression of the HeLa cell through its life cycle is accompanied by centriolar replication and pericentriolar changes that are in synchrony with DNA synthesis and mitosis. The first signs of preparation for replication occur during G₁ at which time the two orthogonal centrioles separate. Replication by budding begins at/or near the initiation of DNA synthesis and is completed by G₂. Pericentriolar changes which probably are causally related to spindle tubule formation occur at this time and include the appearance of vesicles, electron-opaque bodies, and an amorphous pericentriolar halo. These phenomena begin to disappear by late prophase, and the remainder of mitosis manifests decreasing centriolar and pericentriolar activity.