The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes - PubMed (original) (raw)

The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

Christopher B O'Connell et al. J Cell Biol. 2008.

Abstract

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

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Figures

Figure 1.

Figure 1.

Spindle and kinetochore morphology of HeLa MUG. (A and B) Differential interference contrast (A) and centrin1-GFP (B) images of a fixed cell during MUG metaphase. (C–F) This cell was processed for correlative EM. (C) A single lower magnification section through the spindle. Note that chromatin is largely absent from the spindle except for a few small fragments (arrows). (D–F) 70-nm-thick serial sections through a kinetochore. Microtubules attach to the kinetochore end-on as well as laterally (E, arrow).

Figure 2.

Figure 2.

Geometries of kinetochore attachment in MUG. (A) Cells undergoing MUG were fixed and stained for chromatin, microtubules, and kinetochores. Images are maximum intensity projections of deconvolved z slices. Insets (single z slices) illustrate the types of microtubule–kinetochore attachments achievable in MUG: (1) merotelic, (2) monotelic, and (3) lateral. (B) Comparison of kinetochore attachments to spindle poles in normal mitosis versus MUG. Amphitelic or syntelic arrangements are not applicable (N.A.) for unpaired kinetochores.

Figure 3.

Figure 3.

Behavior of unpaired kinetochores. (A) MUG in HeLa cells stably expressing CENP-A–GFP followed by combinational differential interference contrast (top) and three-dimensional fluorescence microscopy (bottom). Kinetochores are extremely dynamic, exhibiting rapid poleward and away from pole movements with a mean velocity of 1.8 ± 0.8 μm/min. Anaphase in this cell (56 s) is marked by cessation of kinetochore dynamics, spindle shortening (compare 49 s with 56 s), and outward movement of chromatin (arrowheads). Not all kinetochores congress before anaphase (arrow). (B) Tracking of a single kinetochore (arrowheads) in the cell shown in A. This as well as many other kinetochores exhibits rapid back and forth movement toward and away from the spindle equator. Time is given in hours/minutes. Bar, 2.5 μm.

Figure 4.

Figure 4.

Evidence of a robust SAC during MUG. (A) HeLa cells undergoing MUG were treated with the indicated drugs and monitored by time-lapse microscopy. The disappearance of the nucleolus (arrowheads) indicates entry into MUG at t = 0. In the presence of nocodazole, there is a prolonged checkpoint arrest followed by escape that is marked by formation of micronuclei and respreading. Exit from MUG is also delayed when cells are treated with monastrol or taxol. Bar, 10 μm. (B) A cell in MUG after washout (t = 0) of 1.5 μM nocodazole. A bipolar spindle forms, and kinetochores congress to the equator (36 s). Soon after, the cell exits MUG, demonstrating satisfaction of the SAC. Time is given in hours/minutes.

Figure 5.

Figure 5.

Behavior of Mad2 and BubR1 during MUG. (A) Cells in MUG were treated with nocodazole to generate unattached kinetochores or were left untreated. Differential interference contrast and fluorescence images of cells stained for DNA, kinetochores, and Mad2. The histogram presents the integrated intensity of Mad2 normalized to nocodazole-treated cells. (B) The distribution of BubR1 during normal mitosis and MUG. The histogram presents the integrated intensity of BubR1 on control and MUG kinetochores normalized to prometaphase. Error bars indicate SEM.

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