Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.     

Nuclear texture in mitotic cells

Nuclear texture in mitotic cells of Drosophila melanogaster imaginal discs has been studied. The distribution of voxels of DAPI fluorescence intensity was used as a quantitative measure of the nuclear texture. The integral characteristics, such as the portion of voxels with a given fluorescent signal level and autocorrelation of pixel intensities, were used. We showed that the nuclear texture specifically changed at various mitotic stages. It can be used for more precise staging of mitosis. Colchicine treatment induced substantial changes in the nuclear texture. This gives a possibility to detect pathologies related to abnormal mitoses by the nuclear-texture approach.     

Nuclear texture in mitotic cells

Changes of nuclear texture in mitotic cells of Drosophila melanogaster imaginal discs were studied. The distribution of voxels DAPI fluorescence intensities was used as the quantitative measure of the nuclear texture. The integral characteristics such as the portion of voxels with a given fluorescent signal level and autocorrelation of pixel intensities were used. We showed the nuclear texture has specific changes at different mitotic stages and this can be used for more precise staging of mitosis. Colchicines treatment pathologies, connected to abnormal mitoses, by nuclear-texture approach.     

Axin localizes to mitotic spindles and centrosomes in mitotic cells

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.     

Hyper-mitogenic drive coexists with mitotic incompetence in senescent cells

When the cell cycle is arrested, even though growth-promoting pathways such as mTOR are still active, then cells senesce. For example, induction of either p21 or p16 arrests the cell cycle without inhibiting mTOR, which, in turn, converts p21/p16-induced arrest into senescence (geroconversion). Here we show that geroconversion is accompanied by dramatic accumulation of cyclin D1 followed by cyclin E and replicative stress. When p21 was switched off, senescent cells (despite their loss of proliferative potential) progressed through S phase, and levels of cyclins D1 and E dropped. Most cells entered mitosis and then died, either during mitotic arrest or after mitotic slippage, or underwent endoreduplication. Next, we investigated whether inhibition of mTOR would prevent accumulation of cyclins and loss of mitotic competence in p21-arrested cells. Both nutlin-3, which inhibits mTOR in these cells, and rapamycin suppressed geroconversion during p21-induced arrest, decelerated accumulation of cyclins D1 and E and decreased replicative stress. When p21 was switched off, cells successfully progressed through both S phase and mitosis. Also, senescent mouse embryonic fibroblasts (MEFs) overexpressed cyclin D1. After release from cell cycle arrest, senescent MEFs entered S phase but could not undergo mitosis and did not proliferate. We conclude that cellular senescence is characterized by futile hyper-mitogenic drive associated with mTOR-dependent mitotic incompetence.     

Stepwise effects of cytokinin activity and DNA synthesis upon mitotic cycle events in partially synchronized tobacco cells

Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period. Although continuous presence of the cytokinin in the culture medium during this lag period was essential to division, cytokinin was not required during mitosis itself. For each cell generation, cytokinin-dependent events are thus completed before mitosis occurs.Two experiments suggested that these cytokinin-dependent events are independent of DNA synthesis:

1. (i) With or without cytokinin, DNA synthesis proceeded normally in the presence of auxin, for at least the time required for one cell generation in complete medium.

2. (ii) In the presence of cytokinin, when DNA synthesis in the lag period was inhibited by FUdR, one normal cell division occurred when cytokinin was withdrawn and DNA synthesis restored by thymidine addition.

In cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.     

Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides

Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0°C or 30°C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0°C and at 30°C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H₂O₂ did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides.     

Chromosomal passengers: the four-dimensional regulation of mitotic events

Chromosomal passengers are proteins that are involved in coordinating the chromosomal and cytoskeletal events of mitosis. The passengers are present in cells as a complex with at least four members: Aurora B, a protein kinase; inner centromeric protein, an activation and targeting subunit; Survivin (function unknown) and Borealin (function also unknown). The kinase is activated at the onset of mitosis, at least partly accomplished by regulation of the levels of its constituents. As mitosis progresses, the kinase complex moves to a highly choreographed series of locations in the mitotic cell, activating key substrates at precise locations and specific times. Functions that require chromosomal passenger activity include chromatin modification (phosphorylation of histone H3), correction of kinetochore attachment errors, aspects of the spindle assembly checkpoint, assembly of a stable bipolar spindle and the completion of cytokinesis. The chromosomal passenger complex provides an essential mechanism for mitotic regulation.     

Centromere mitotic recombination in mammalian cells

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.     

Post-translational modifications in mitotic yeast cells

We have recently shown that secretion of invertase is not inhibited in the yeast Saccharomyces cerevisiae during mitosis, but continues, as during interphase. This is in contrast with the mammalian cell, where membrane traffic stops at the onset of prometaphase. Here we extend our findings by showing that the bulk of the cell surface glycoproteins and mannans, as well as the yeast pheromone alpha-factor, traverse the secretory pathway during mitosis. We show that the mitotic cells are able to carry out several types of post-translational modification of secretory proteins. (a) The secretory protein invertase was oligomerized and extensively glycosylated, (b) the N-glycan cores of bulk-cell surface mannans were extended with outer chains, (c) some N-glycans were phosphorylated, (d) the protein-bound O-glycans were extended up to tetramannosides, (e) prepro-ka-factor was proteolytically processed to alpha-factor molecules. We conclude that the secretory pathway in yeast remains fully functional throughout the cell cycle.     

gamma-tubulin plays an essential role in the coordination of mitotic events

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.     

A guide to classifying mitotic stages and mitotic defects in fixed cells

Cell division is fundamental to life and its perturbation can disrupt organismal development, alter tissue homeostasis, and cause disease. Analysis of mitotic abnormalities provides insight into how certain perturbations affect the fidelity of cell division and how specific cellular structures, molecules, and enzymatic activities contribute to the accuracy of this process. However, accurate classification of mitotic defects is instrumental for correct interpretation of data and formulation of new hypotheses. In this article, we provide guidelines for identifying specific mitotic stages and for classifying normal and deviant mitotic phenotypes. We hope this will clarify confusion about how certain defects are classified and help investigators avoid misnomers, misclassification, and/or misinterpretation, thus leading to a unified and standardized system to classify mitotic defects.     

Cyclin B is associated with centrosomes in Drosophila mitotic cells.

We have studied by way of confocal laser scanning microscopy the subcellular localization of cyclin B in Drosophila-cultured cells and report here evidence that a part of the cyclin B cell pool is closely associated with the centrosome. This cyclin B centrosomal signal is strong in prophase and metaphase but disappears during anaphase. Moreover, the signal is absent in the acentriolar Drosophila cell line 1182-4. These results put forward additional arguments suggesting that the centrosome plays an important role in the control of the cell cycle.     

Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells

Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re- initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.     

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.