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

1994 ◽  
Vol 127 (5) ◽  
pp. 1301-1310 ◽  
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.

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

1979 ◽  
Vol 80 (3) ◽  
pp. 674-691 ◽  
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.

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

PROTOPLASMA ◽  
2001 ◽  
Vol 218 (3-4) ◽  
pp. 192-202 ◽  
Author(s):  
J. F. Giménez-Abián ◽  
D. J. Clarke ◽  
M. I. Giménez-Abián ◽  
C. de la Torre ◽  
G. Giménez-Martín
Keyword(s):  
Nuclear Envelope ◽  
Multinucleate Cells ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset

scholarly journals Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

1998 ◽  
Vol 142 (4) ◽  
pp. 1013-1022 ◽  
Author(s):  
Conly L. Rieder ◽  
Richard W. Cole
Keyword(s):  
Cell Cycle ◽  
Somatic Cells ◽  
Early Prophase ◽  
Checkpoint Control ◽  
Late Prophase ◽  
Cytoplasmic Microtubule ◽  
Nuclear Envelope Breakdown ◽  
Prophase Nucleus ◽  
Nuclear Events ◽  
A Cell

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

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

1996 ◽  
Vol 135 (1) ◽  
pp. 201-214 ◽  
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.

scholarly journals F-actin patches nucleated on chromosomes coordinate capture by microtubules in oocyte meiosis

2018 ◽  
Author(s):  
Mariia Burdyniuk ◽  
Andrea Callegari ◽  
Masashi Mori ◽  
François Nédélec ◽  
Péter Lénárt
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cells ◽  
Oocyte Nucleus ◽  
Chromosome Loss ◽  
Dependent Manner ◽  
Actin Network ◽  
Nuclear Envelope Breakdown ◽  
Oocyte Meiosis ◽  
Spindle Microtubules ◽  
Ran Gtp

AbstractCapture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-μm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin patches form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of these F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary, as early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

Probing The Mechanisms Underlying Kinetochore Behavior In Vertebate Cells Using Combinations of Advanced Light and 3-D Electron Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 217-218
Author(s):  
B. F. McEwen ◽  
A.B. Heagle ◽  
C.L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Attachment Site ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Daughter Cells ◽  
Primary Constriction ◽  
Sister Kinetochore ◽  
To Receive ◽  
Chromosome Motion

For daughter cells to receive equal copies of the genome during mitosis, the replicated chromosomes must attach to and move bi-directionally on the mitotic spindle. A chromosome becomes attached to the spindle via a pair specialized structures, known as kinetochores, that are positioned on opposite sides of its primary constriction (one on each of the two chromatids). In addition to being the spindle attachment site, kinetochores are also involved in producing and/or transmitting the forces for chromosome motion. In vertebrates the kinetochore closest to a spindle pole at the time of nuclear envelope breakdown usually is the first to attach to the spindle. As a result of this attachment the now “monooriented” chromosome moves toward the closest pole where its only attached kinetochore initiates oscillatory motions toward and away from that pole until the unattached sister kinetochore acquires microtubules (Mts) from the opposite spindle pole.

scholarly journals Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.

1995 ◽  
Vol 129 (6) ◽  
pp. 1447-1458 ◽  
Author(s):  
G Sluder ◽  
E A Thompson ◽  
C L Rieder ◽  
F J Miller
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Control Mechanisms ◽  
Checkpoint Control ◽  
Intact Male ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Sperm Nuclei ◽  
Histone Kinase

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.

scholarly journals F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis

2018 ◽  
Vol 217 (8) ◽  
pp. 2661-2674 ◽  
Author(s):  
Mariia Burdyniuk ◽  
Andrea Callegari ◽  
Masashi Mori ◽  
François Nédélec ◽  
Péter Lénárt
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cells ◽  
Oocyte Nucleus ◽  
Chromosome Loss ◽  
Dependent Manner ◽  
Actin Network ◽  
Nuclear Envelope Breakdown ◽  
Oocyte Meiosis ◽  
Spindle Microtubules ◽  
Ran Gtp

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin–driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin “patches” form around chromosomes in a Ran-GTP–dependent manner, and we propose that these structures sterically block kinetochore–microtubule attachments. Once F-actin–driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin–driven transport leading to chromosome loss and formation of aneuploid eggs.

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

2010 ◽  
Vol 38 (1) ◽  
pp. 72-77 ◽  
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.

scholarly journals Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly

2009 ◽  
Vol 184 (3) ◽  
pp. 365-372 ◽  
Author(s):  
Alberto Toso ◽  
Jennifer R. Winter ◽  
Ainslie J. Garrod ◽  
Ana C. Amaro ◽  
Patrick Meraldi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Envelope ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Aurora A ◽  
Bipolar Spindle ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Centrosome Separation ◽  
Dependent Pathway

In animal somatic cells, bipolar spindle formation requires separation of the centrosome-based spindle poles. Centrosome separation relies on multiple pathways, including cortical forces and antiparallel microtubule (MT) sliding, which are two activities controlled by the protein kinase aurora A. We previously found that depletion of the human kinetochore protein Mcm21RCENP-O results in monopolar spindles, raising the question as to whether kinetochores contribute to centrosome separation. In this study, we demonstrate that kinetochores promote centrosome separation after nuclear envelope breakdown by exerting a pushing force on the kinetochore fibers (k-fibers), which are bundles of MTs that connect kinetochores to centrosomes. This force is based on poleward MT flux, which incorporates new tubulin subunits at the plus ends of k-fibers and requires stable k-fibers to drive centrosomes apart. This kinetochore-dependent force becomes essential for centrosome separation if aurora A is inhibited. We conclude that two mechanisms control centrosome separation during prometaphase: an aurora A–dependent pathway and a kinetochore-dependent pathway that relies on k-fiber–generated pushing forces.

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