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Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis.

We have discovered a novel mitosis-specific human autoantigen that arises at the centromeres of prophase chromosomes, but ultimately participates in formation of an organelle that bisects the cell at late anaphase and during telophase. The organelle, discernible as a three-dimensional disc by confocal microscopy, encompasses the entire midzone diameter, and its distribution survives disassembly of interpolar microtubules by cold temperature treatment and detergent lysis of cells. Cytokinetic furrow contraction proceeds normally in dihydrocytochalasin B (DCB)-treated cells, and antigen distribution in the furrow is unaltered. In DCB, the furrow retracts in early interphase, coincident with loss of normal membrane association with the disc, resulting in the formation of binucleate cells. The midzone disc in both drug-treated and normal cells is present at the correct time and position to play a central role in cytokinesis. By immunocytochemistry, the disc appears to contain myosin but not actin. The position of the disc and the possible presence of myosin suggest that cytokinesis may involve the interaction of the disc organelle with actin in the cell cortex to produce cleavage in mammalian cells.

Actins

The telophase disc: its possible role in mammalian cell cleavage.

The molecular signals that determine the position and timing of the furrow that forms during mammalian cell cytokinesis are presently unknown. It is apparent, however, that these signals are generated by the mitotic spindle after the onset of anaphase. Recently we have described a structure that bisects the cell during telophase at the position of the cytokinetic furrow. This structure, the telophase disc, appears to be templated by the mitotic spindle during anaphase, and precedes the formation of the cytokinetic furrow. The relationship of the telophase disc to the myosin and actin based furrowing mechanism is discussed here. We propose that the telophase disc may determine the position and timing of cleavage by recruitment and alignment of myosin.

Actins

Anaphase-telophase analysis of chromosomal damage induced by chemicals.

Three main aspects involved in the chemical induction of anaphase-telophase aberrations in the first mitosis after treatment were analyzed: 1) the relationship between the frequency of anaphase-telophase aberrations and the time of fixation after treatment; 2) the dose-response relationships; and 3) the proliferative rate of cells exposed to chemicals which interact with DNA by different mechanisms. Experiments were carried out using Chinese hamster ovary (CHO) cells. The compounds examined were adriamycin (ADR) and mitomycin C (MMC). The frequency of cells with chromatin bridges or with lagging chromosomes as well as the mitotic index was determined in each experiment. The results obtained showed that 1) chromatin bridges and lagging chromosomes are apparently induced during the S period of the previous interphase; 2) the increase in the cytotoxicity index (inferred from the mitotic index) and the frequency of cells with chromatin bridges and lagging chromosomes were proportional to the treatment lapse and to the dose employed; and 3) the effect of ADR on cell growth differs from the effect of MMC. While ADR decreased the mitotic activity of cells in logarithmic growth phase, MMC induced mitotic delay. In accordance with these results, the occurrence of chromatin bridges in anaphase-telophase could be explained by the induction of chromosome stickiness and, to a lesser extent, by the induction of exchange-type aberrations. On the other hand, lagging chromosomes seem to be the result of chromatid or chromosome breaks because the lagging chromosomes observed were primarily, if not all, fragments and not whole chromosomes. Our evaluation of the anaphase-telophase test indicates that it is very sensitive method for the detection of chemical clastogens, but other factors, such as mitotic depression, must be taken into account to avoid false-negative results.

Anaphase

The Swi5 transcription factor of Saccharomyces cerevisiae has a role in exit from mitosis through induction of the cdk-inhibitor Sic1 in telophase.

Deactivation of the B cyclin kinase (Cdc28/Clb) drives the telophase to G1 cell cycle transition. Here we investigate one of the control pathways than contributes to kinase deactivation, involving the cell cycle-regulated production of the cdk inhibition Sic1. We show that the cell cycle timing of SIC1 expression depends on the transcription factor Swi5, and that Swi5-dependent SIC1 expression begins during telophase. In contrast to Swi5, the related transcription factor Ace2, which can also induce SIC1 expression, is not active during telophase. The functional consequence of Swi5-regulated SIC1 expression in vivo is that both sic1 delta and swi5 delta strains have identical mitotic exit-related phenotypes. First, both are synthetically lethal with dbj2 delta, resulting in cell cycle arrest in telophase. Second, both are hypersensitive to overexpression of the B cyclin CLB2. Thus Swi5-dependent activation of the SIC1 gene contributes to the deactivation of the B cyclin kinase, and hence exit from mitosis.

CDC28 Protein Kinase, S cerevisiae

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.

Anaphase

Staurosporine induces telophase arrest and apoptosis blocking mitosis exit in human Chang liver cells.

Staurosporine is a protein kinase inhibitor that is known to block G1/S and G2/M cell cycle transitions, resulting in the accumulation of G1 or G2 interphase cells, depending on the applied dosage. It is, however, not known to block mitosis (M-phase) progression. We show here that a 15 min prepulse with 500 nM staurosporine had caused mitotic cell arrest in human Chang liver cells, accumulating telophase cells distinguished by their doublet nuclei and cytokinetic constrictions. The arrested telophase cells perished by apoptosis. Telophase-specific cell cycle arrest and apoptosis are novel. Staurosporine targeting cytokinesis could provide insights into late mitosis where control mechanisms remain largely unknown.

Apoptosis

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase.

. The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1-2 microm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.

Anaphase

Effect of divalent cations and chelators on metaphase to telophase progression and nuclear envelope formation in Chinese hamster cells.

Chinese hamster DON cells in log phase were treated with Colcemid in the G2 period with or without divalent cation chelating agents. The metaphase cells were isolated and incubated in two ways: 1) without Colcemid but with chelating agents or La3+ and observed for metaphase to telophase progression, and 2) with Colcemid, with or without chelating agents and the rate of micronuclei formation in the absence of anaphase monitored. The effect of the chelating agents on cellular 45Ca2+ during metaphase to telophase progression was also studied. The results indicate that Ca2+ and possibly Mg2+ ions are involved in the regulation of certain segments of mitosis. The reduction of environmental and plasma membrane associated Ca2+ with the chelators and La3+ promoted the metaphase to telophase progression as well as nuclear envelope and micronuclei formation.

Animals

Interzone microtubule behavior in late anaphase and telophase spindles.

We have studied microtubule behavior in late anaphase and telophase spindles of PtK1 cells, using fluoresceinated tubulin (DTAF-tubulin), microinjection, and laser microbeam photobleaching. We present the results of two novel tests which add to the evidence that DTAF-tubulin closely mimics the behavior of native tubulin in vivo. (a) Microinjected DTAF-tubulin was as effective as injected native tubulin in promoting division of taxol-dependent mitotic mutant cells that had been deprived of taxol. (b) Microinjected colchicine-DTAF-tubulin complex was similar to injected colchicine-native tubulin complex in causing depolymerization of spindles. Immediately after microinjection of DTAF-tubulin into wild-type cells during late anaphase or telophase, fluorescence incorporation by microtubules was seen in chromosomal half-spindles and just behind the chromosomes, but there was no fluorescence incorporation near the middle of the interzone. Over the next few minutes, tubulin fluorescence accumulated at the center of the interzone (the equator), becoming progressively more intense. In other experiments, cells were microinjected with DTAF-tubulin at prophase and allowed to equilibrate for 30 min. Cells that had progressed to late anaphase were then photobleached to reduce the fluorescence in the central portion of the interzone. Over a period of several minutes, the only substantial redistribution of fluorescence was the appearance of a bright area at the equator of the interzone. Both the site of fluorescence incorporation and the photobleaching data suggest that tubulin adds to the elongating spindle interzone near the equator where the plus ends of the interdigitated microtubules are located. In further experiments, several dark lines were photobleached perpendicular to the pole-to-pole axis of fluorescent anaphase-telophase spindles. Time-dependent changes in the spacings between the lines indicated that the two halves of the interzone lying on opposite sides of the spindle equator moved away from one another. This shows that the interdigitated microtubules, which make up most of the interzone, can undergo antiparallel sliding. Our data support a model for anaphase B in which plus end elongation of interdigitated microtubules and antiparallel sliding contribute to chromosome separation.

Anaphase

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery.

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.

Actins

The STUD gene is required for male-specific cytokinesis after telophase II of meiosis in Arabidopsis thaliana.

During male meiosis in wild-type Arabidopsis the pollen mother cell (PMC) undergoes two meiotic nuclear divisions in the absence of cell division. Only after telophase II is a wall formed which partitions the PMC into four microspores. Each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. In this paper we describe the isolation and the phenotypic characterization of mutations in the STUD (STD) gene, which is specifically required for male-specific cytokinesis after telophase II of meiosis. Although the male meiotic nuclear divisions are normal in std mutant plants, no walls are formed resulting in a tetranucleate microspore. Despite the absence of cell division in the PMC, postmeiotic development in the coenocytic microspore proceeds relatively normally, resulting in the formation of large pollen grains which contain four vegetative nuclei and up to eight sperm cells. Interestingly, these enlarged pollen grains which contain multiple vegetative nuclei and extra sperm cells behave as single male gametophytes, producing only single pollen tubes and resulting in partial male fertility in std mutant plants. Characterization of the process of pollen development and pollen function in std mutants thus reveals two different types of developmental regulation. Each of the four nuclei found in a std microspore following meiosis is capable of independently undergoing the complete mitotic cell division (including cytokinesis) which the single nucleus of a wild-type microspore would normally undertake. The ability of the four meiotic products to independently continue through mitosis does not depend on their division into separate cells, but is controlled by some subcellular component found within the coenocytic microspore. By contrast, the mature std pollen grain functions as a unit and produces only a single pollen tube despite the presence of multiple nuclei within the vegetative cell, suggesting that this process is controlled at the cellular level independently of the extra subcellular components.

Arabidopsis

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Animals

Naphthofurans induced chromosomal aberrations detected in metaphase, anaphase and telophase V79 Chinese hamster cells.

The mutagenic activities of 5 newly synthesized naphthofurans were analysed in two in vitro cytogenetic assays: the metaphase chromosomal aberration assay and the anaphase telophase bridge-fragment assay. Both assays were conducted using V79 Chinese hamster cells. The compounds included: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitro-naphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The cells were treated with 3 concentrations (0.1, 0.2 and 0.4 microgram/ml) of each compound, in the dose range already tested in studies on the mutagenic properties of the same compounds realised with other systems. The highest concentration, only, was used in the anaphase-telophase assay. In the first approach, compounds A, B and C were active while compounds D and E did not increase significantly the aberration frequency above that of the DMSO controls. The results were confirmed in the second approach. They demonstrated that the two studies were complementary. Based on their genotoxic activities, the 5 compounds were ranked in the following decreasing order of potency: A congruent to B much greater than C greater than D congruent to E congruent to DMSO; which is comparable to the ranking order obtained in different in vitro mutagenic and carcinogenic assays. All these activities are closely related to the highly specific molecular structure of each compound, particularly to the nature and position of the different substituents introduced on the skeleton.

Anaphase

Allium cepa anaphase-telophase root tip chromosome aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azide, and ethyl methanesulfonate.

The Allium anaphase-telophase assay was used to show genotoxicity of N-methyl-N-nitrosourea (MNU), maleic hydrazide (MH), sodium azide (NaN3) and ethyl methanesulfonate (EMS). All agents induced chromosome aberrations at statistically significant levels. The rank of the lowest doses with positive effect was as follows: NaN3 0.3 mg/l < MH 1 mg/l < MNU 41 mg/l < EMS 100 mg/l. The results were compared with results from other plant assays (Arabidopsis, Vicia, Tradescantia) and for MH and MNU the values were found to be within the same range, whereas the results in the Allium test for NaN3 and EMS were in a lower range than that found for the other plant assays. EMS and MMS (methyl methanesulfonate), two chemicals used as positive controls in mutagenicity testing, were compared in the Allium test, and MMS was found to be about ten times more potent in inducing chromosome aberrations than EMS. Recording of micronuclei in interphase cells showed that this endpoint does not give more information of clastogenicity than recording of chromosome aberrations in anaphase-telophase cells.

Allium

The nucleolus in telophase, interphase and prophase.

The ultrastructure of telophase to interphase has been followed in a green alga, Spirogyra submargaritata. A series of changes transitional between the late anaphase chromatid, the decondensing chromatid of telophase, and the 'pale fibrillar material' occupying channels in the nucleolus at interphase have been demonstrated. Early stages in the regeneration of the nucleolus are described. It has been shown that the pale fibrillar material in the nucleolus is attached to, and continuous with, the fully condensed (chromocentric) part of the nucleolar-organizing chromosome at interphase. It is also shown that in early prophase, the channels in the nucleolonema of the nucleolus are no longer occupied by pale fibrillar material, but instead a long section of condensed chromosome is present, traversing the nucleolonema. It is contended that these observations taken together constitute evidence that the pale fibrillar material of the nucleolus is the chromatin of the nucleolar-organizing region of the chromosome, expanded for transcription. A model of the nucleolus as it is seen in most electron-microscope sections, and as it can be interpreted in the light of present-day knowledge about it, is presented. A brief review of the relevant literature considers the views supporting the mode, and the contrary views, implicating the use of the term 'nucleolar organizer', that are still current at the present time.

Cell Nucleolus

Evaluation of the Allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater.

The Allium anaphase-telophase test was evaluated to find out if it could be recommended in the screening of wastewater for genotoxicity. Five mutagenic or carcinogenic chemicals usually found in wastewater were tested in the Allium anaphase-telophase test. Sodium dichromate (25 microM), benzene (100 microM), dichloromethane (175 microM) and 1,1,1-trichloromethane (175 microM) increased the frequency of chromosome aberrations in the root cells, whereas formaldehyde (1 mM) was found to be non-mutagenic in this test system. Other studies where chemicals were tested in the Allium test were reviewed. For 15 chemicals the results were compared with results from the Ames test, the Microscreen assay, and carcinogenicity tests in rodents. The sensitivity of the Allium test was calculated to be 82%. In conclusion the Allium test is recommended for the screening of wastewater because it has a high sensitivity, is cheap, rapid, easy to handle, and because it can be used on wastewater without pretreatment of the sample.

Allium

[Electron microscopic study of the nuclear membrane of SPEV cells during mitosis. II. Reconstruction of the nuclear membrane (anaphase and telophase)].

Reformation of the nuclear envelope of the PKLV cells starts in the early anaphase when numerous contacts between membranous elements and chromosomes become visible. The nuclear envelope reforms, first, on the periphery of chromosomes and in chromosome's centromeric regions, and later - in the telomeric regions. The latest reconstruction of nuclear envelope occurs in spaces where microtubules come up close to the "nuclei", The appearance of normal pore complexes in the late telophase is preceded by the appearance, in the early and middle telophase, of pores with unusual structure.

Cell Line