Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells

J. F. Giménez-Abián; D. J. Clarke; M. I. Giménez-Abián; C. de la Torre; G. Giménez-Martín

doi:10.1007/bf01306608

Role of spindle microtubules in the control of cell cycle timing.

The Journal of Cell Biology ◽

10.1083/jcb.80.3.674 ◽

1979 ◽

Vol 80 (3) ◽

pp. 674-691 ◽

Cited By ~ 66

Author(s):

G Sluder

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Sea Urchin ◽

Control Cell ◽

Microtubule Assembly ◽

Nuclear Envelope Breakdown ◽

Anaphase Onset ◽

Spindle Cells ◽

Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

Download Full-text

Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis.

The Journal of Cell Biology ◽

10.1083/jcb.135.1.201 ◽

1996 ◽

Vol 135 (1) ◽

pp. 201-214 ◽

Cited By ~ 133

Author(s):

Y Zhai ◽

P J Kronebusch ◽

P M Simon ◽

G G Borisy

Keyword(s):

Nuclear Envelope ◽

Fluorescence Ratio ◽

Subsequent Increase ◽

Abrupt Decrease ◽

Nuclear Envelope Breakdown ◽

Anaphase Onset ◽

Chromosome Attachment ◽

Direct Fluorescence ◽

Cytoplasmic Microtubules ◽

Tubulin Immunofluorescence

We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results showed no net decrease in polymer, indicating that the disassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level. We now examine the reverse process, the interphase-mitosis transition. Specifically, we quantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakdown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti-tubulin immunofluorescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis. Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple redistribution of tubulin at constant polymer level as the mitosis-interphase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.

Download Full-text

Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1301 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1301-1310 ◽

Cited By ~ 319

Author(s):

C L Rieder ◽

A Schultz ◽

R Cole ◽

G Sluder

Keyword(s):

Nuclear Envelope ◽

Somatic Cells ◽

Checkpoint Control ◽

Average Interval ◽

Nuclear Envelope Breakdown ◽

Anaphase Onset ◽

Sister Kinetochore ◽

Chromosome Motion

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.

Download Full-text

Cyclin A and Nek2A: APC/C–Cdc20 substrates invisible to the mitotic spindle checkpoint

Biochemical Society Transactions ◽

10.1042/bst0380072 ◽

2010 ◽

Vol 38 (1) ◽

pp. 72-77 ◽

Cited By ~ 19

Author(s):

Wouter van Zon ◽

Rob M.F. Wolthuis

Keyword(s):

Nuclear Envelope ◽

Spindle Checkpoint ◽

Cyclin A ◽

Cyclin B1 ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Nuclear Envelope Breakdown ◽

Anaphase Onset ◽

Sister Chromatids ◽

Spindle Microtubules

Active cyclin B1–Cdk1 (cyclin-dependent kinase 1) keeps cells in mitosis, allowing time for spindle microtubules to capture the chromosomes and for incorrect chromosome-spindle attachments to be repaired. Meanwhile, securin, an inhibitor of separase, secures cohesion between sister chromatids, preventing anaphase onset. The spindle checkpoint is a signalling pathway emerging from improperly attached chromosomes that inhibits Cdc20, the mitotic activator of the APC/C (anaphase-promoting complex/cyclosome) ubiquitin ligase. Blocking Cdc20 stabilizes cyclin B1 and securin to delay mitotic exit and anaphase until all chromosomes reach bipolar spindle attachments. Cells entering mitosis in the absence of a functional spindle checkpoint degrade cyclin B1 and securin right after nuclear-envelope breakdown, in prometaphase. Interestingly, two APC/C substrates, cyclin A and Nek2A, are normally degraded at nuclear-envelope breakdown, even when the spindle checkpoint is active. This indicates that the APC/C is activated early in mitosis, whereas cyclin B1 and securin are protected as long as the spindle checkpoint inhibits Cdc20. Remarkably, destruction of cyclin A and Nek2A also depends on Cdc20. The paradox of Cdc20 being both active and inhibited in prometaphase could be explained if cyclin A and Nek2A are either exceptionally efficient Cdc20 substrates, or if they are equipped with ‘stealth’ mechanisms to effectively escape detection by the spindle checkpoint. In the present paper, we discuss recently emerging models for spindle-checkpoint-independent APC/C–Cdc20 activity, which might even have implications for cancer therapy.

Download Full-text