Ultraviolet-Microbeam Irradiation of Newt-Cell Cytoplasm: Spindle Destruction, False Anaphase, and Delay of True Anaphase
TL;DR: Mitotic tissue-culture cells of the newt Triturus viridescens were irradiated in various cytoplasmic regions with monochromatic or heterochromatics ultraviolet microbeams to generate new cells that secrete nonvolatile substance which acts as a “spatially aggregating substance” to reprogram the cell wall.
Abstract: Mitotic tissue-culture cells of the newt Triturus viridescens were irradiated in various cytoplasmic regions with monochromatic or heterochromatic ultraviolet microbeams (conical, with 25° half-angle, 8 μ in diameter in focal plane). Depending on exposure, some spindles were destroyed (i.e., true anaphase was prevented), and this was sometimes followed by quasirosette formation and false anaphase. Efficiency of spindle destruction was not detectably affected by the presence of a centrosome or part of the formed metaphase spindle in the irradiated region. For 280-nm irradiation the exposure which destroyed 50% of spindles was $2\times 10^{12}$ in terms of incident photons per cell times the cell depth in microns. Direct irradiation of centrosomes did not impair their mutual attraction for kinetochores. In metaphase-irradiated cells which survived spindle destruction, delay of true anaphase was critically dependent on whether irradiation occurred before or after the arrival of the last kinetochore at the sp...
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TL;DR: It appears that some checkpoints are eliminated during the early embryonic development of some organisms; this fact may pose special problems for the fidelity of embryonic cell division.
Abstract: The events of the cell cycle of most organisms are ordered into dependent pathways in which the initiation of late events is dependent on the completion of early events. In eukaryotes, for example, mitosis is dependent on the completion of DNA synthesis. Some dependencies can be relieved by mutation (mitosis may then occur before completion of DNA synthesis), suggesting that the dependency is due to a control mechanism and not an intrinsic feature of the events themselves. Control mechanisms enforcing dependency in the cell cycle are here called checkpoints. Elimination of checkpoints may result in cell death, infidelity in the distribution of chromosomes or other organelles, or increased susceptibility to environmental perturbations such as DNA damaging agents. It appears that some checkpoints are eliminated during the early embryonic development of some organisms; this fact may pose special problems for the fidelity of embryonic cell division.
3,048 citations
TL;DR: Results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy, and that p53-deficient mouse embryos were exposed to spindle inhibitors.
Abstract: Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.
695 citations
TL;DR: Tension stabilizes the proper chromosome configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.
Abstract: When cells divide, the chromosomes must be delivered flawlessly to the daughter cells. Missing or extra chromosomes can result in birth defects and cancer. Chance events are the starting point for chromosome delivery, which makes the process prone to error. Errors are avoided by diverse uses of mechanical tension from mitotic forces. Tension stabilizes the proper chromosome configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.
579 citations
TL;DR: This work applies tension to an improperly attached chromosome with a micromanipulation needle and finds that the entry into anaphase and the completion of mitosis was delayed by 5–6 hours when the misattached chromosome was placed under tension.
Abstract: Every time a cell divides, the chromosomes must be distributed accurately to the daughter cells. Errors in distribution arise if chromosomes are improperly attached to the mitotic spindle. Improper attachment is detected by a cell-cycle checkpoint in many cells and the completion of cell division is delayed, allowing time for error correction. How is an improperly attached chromosome detected? An absence of tension from mitotic forces is one possibility. Here we test this possibility directly by applying tension to an improperly attached chromosome with a micromanipulation needle. In the absence of tension, the entry into anaphase and the completion of mitosis was delayed by 5-6 hours. When the misattached chromosome was placed under tension, however, the cell entered anaphase in 56 minutes, on average. Tension from mitotic forces or from a micromanipulator's needle evidently signals to the checkpoint that all is in order and that cell division can proceed.
573 citations
TL;DR: It is concluded that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle, and that the chromosomes move poleward during anaphase.
Abstract: To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
562 citations
Cites background from "Ultraviolet-Microbeam Irradiation o..."
...Indeed, Zirkle (1970) found no correlation between when the last pair of congressing kinetochores reached the spindle equator and the duration of metaphase-a phase of mitosis that cannot be estimated quantitatively....
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...If the metaphase-anaphase checkpoint is relieved when the last monooriented chromosome biorients (as proposed e.g., by Zirkle 1970, Mclntosh 1991) , then the cell should enter anaphase after a relatively constant interval from when the last monooriented chromosome attaches in a bipolar fashion to…...
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...Based on cine records Zirkle (1970) concluded that "newt cells, like most others, never start anaphase" in the presence of monooriented chromosomes ....
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...Previous attempts to determine the influence of monooriented chromosomes on the timing of anaphase onset (i.e., Zirkle, 1970) relied on estimating the duration of "metaphase ;' which is traditionally defined to start when all of the kinetochores are positioned on the spindle equator....
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TL;DR: A new and widely applicable method with which considerably smaller fractions of average-sized cells can be bombarded, and mention of some of the results obtained.
Abstract: SELECTIVE EXPOSURE of fractions of intact individual cells to high-energy radiation is of interest fo r two reasons. First, i t has obvious use in radiobiology as a means of gaining information about the mechanisms by which radiations produce their strikingly injurious effects on living systems. Second, and of much more general application, it can aid in analyzing the normal functions of the various cell parts by selectively altering them. I n this respect it complements microsurgery. Irradiation of parts of single intact cells has been done before, both with ultraviolet light (1)and with high-energy radiation (2-7) ,3 but in each instance the success of the technique has been largely dependent on some peculiarity of cell structure, e.g., exceptionally large size (2, 7) or a n eccentric nucleus (2, 3), and accordingly has been quite'limited in application. Also the cell fraction irradiated has in most cases been quite large (25-50 per cent). This paper contains a brief description of a new and widely applicable method with which considerably smaller fractions of average-sized cells can be bombarded, and mention of some of the results obtained.
221 citations