Kinetochore stretching inactivates the spindle assembly checkpoint

Abstract

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.

Figure 1.

Kinetochores undergo intermittent deformations. (A) Measurement of intra- and interkinetochore lengths and distances. The green and red circles represent EGFP–CENP-A and mCherry-Mis12, respectively. (B) Setting a threshold for the intra- and interkinetochore lengths/distances. Fixed cells treated with 160 ng/ml (531.6 nM) nocodazole (Noc.) were analyzed for the intra- and interkinetochore lengths and distances, and their threshold values (red dotted lines) were calculated as 0.10 µm and 0.79 µm, respectively (n = 112 kinetochores from 13 cells; bottom). These values were applied to the profile in a mock condition (n = 118 kinetochores from 12 cells; top). (C) A profile for intra- and interkinetochore lengths and distances obtained for EGFP-Ndc80 and Spc25-mCherry. The threshold values determined in the 160-ng/ml nocodazole condition (n = 24 kinetochores from 5 cells) were 0.11 µm and 0.76 µm for the intra- and interkinetochore length/distance, respectively (blue dotted lines). In a mock condition, <3% of cases had intrakinetochore length beyond the threshold level (n = 38 kinetochores from 5 cells). (D) A kymograph of a kinetochore pair. Note that mCherry-Mis12 signal (red) deviates from EGFP–CENP-A signal (green) transiently. The right panel shows representative intra- and interkinetochore lengths and distances over time. Red diamonds indicate peaks for the intrakinetochore lengths that were subjected to the analysis in E. (E) Basis for CENP-A and Mis12 behavior. Relative poleward positions of CENP-A and Mis12 are determined by averaging 76 cases. Time 0 defines the peak of the intrakinetochore length, and the position of CENP-A at −6 s is set to 0. Arrows indicate the movements of CENP-A and Mis12 during 3-s intervals, and the asterisks mark the significant movement change (t test; *, P < 0.05). Movements of CENP-A and Mis12 on one kinetochore did not significantly affect their movements on the other kinetochore (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200811028/DC1); the stretching occurs on kinetochore sisters independently from each other. (F) A cartoon illustrating kinetochore extension and recoiling. Dotted red and green arrows indicate significant poleward movements of CENP-A and Mis12, respectively. Bar, 1 µm.

Figure 2.

Kinetochore stretching is required to inactivate the SAC. (A) Release of Mad2 from kinetochores in low nocodazole (Noc.) conditions. The kinetochore Mad2 staining in cells treated with the indicated concentrations of nocodazole (left) were classified according to their intensities. A histogram compares the percentages of metaphase cells that have no Mad2 on any kinetochores (white) with cells that have positive (black) or ambiguous (gray) Mad2 staining on at least one kinetochore (n = 16 cells). (B) Enrichment of BubR1 at kinetochores in the low nocodazole conditions. Kinetochore BubR1 intensities were classified into high (positive; black) or low (anaphase level; white). The percentages of metaphase cells for BubR1 staining were summarized in a histogram as in A (n = 16 cells). (C) Suppression of the kinetochore stretching in low nocodazole conditions. Kinetochore profiles were analyzed in the conditions indicated. Red dotted lines represent threshold values defined in Fig. 1 B. The stretched kinetochores were found in 15.5%, 6.9%, and 7.1% of cases, and the interkinetochore distances were 1.19 ± 0.03 µm (mean ± SEM), 1.13 ± 0.03 µm, and 0.88 ± 0.02 µm for mock (n = 71, 9 cells) and 7-ng/ml (n = 72, 7 cells) and 10-ng/ml (n = 56, 6 cells) nocodazole conditions, respectively. (D) Mitotic progression in low nocodazole conditions. The time intervals between the indicated mitotic events were measured in mock (n = 86 cells) or 7-ng/ml (n = 65 cells) or 10-ng/ml (n = 38 cells) nocodazole treatment experiments. Horizontal lines indicate the mean. Bars, 1 µm.

Figure 3.

Suppression of kinetochore stretching by condensin I depletion correlates with a delay in mitotic progression. (A) Depletion of CAP-D2. Cells transfected with the indicated siRNA were analyzed by Western blotting with CAP-D2 antibodies. The Ponceau S staining serves as a loading control. (B) Live cell imaging of condensin I–depleted cells. EGFP–histone H2B–expressing cells were transfected with siRNAs and analyzed after 72 h. Mitosis images were extracted from long-term imaging experiments and aligned on the time axis according to nuclear envelope breakdown (NEBD). The time points for metaphase alignment and anaphase onset are defined by dashed lines. (C) Mitotic progression in condensin I–depleted cells. The time intervals between the indicated mitotic events were measured in control (n = 38 cells) or condensin I RNAi (n = 36 cells) experiments. Horizontal lines indicate the mean. (D) Kinetochore stretching is suppressed in condensin I–depleted cells. Kinetochore profiles were assessed with the threshold values defined in Fig. 1 B (red dotted lines). The incidences for the kinetochore stretching were 16.2% and 4.5%, and the interkinetochore distances were 1.19 ± 0.03 µm (mean ± SEM) and 2.02 ± 0.07 µm for control (n = 74, 9 cells) and condensin I–depleted cells (n = 66, 10 cells), respectively. Bar, 10 µm.

Figure 4.

Kinetochore stretching promotes APC/C activation in metaphase. (A) Metaphase to anaphase transition in control or in 7 ng/ml nocodazole-treated cells. Levels of mCherry–cyclin B1 were followed with Hoechst DNA staining. Images were extracted from long-term imaging experiments and aligned on the time axis according to the metaphase alignment. (B) Kinetics of cyclin B1 proteolysis. Fluorescence intensities of cyclin B1 were measured during metaphase cells from both mock-treated (black line; n = 12) and 7 ng/ml nocodazole-treated experiments (brown line; n = 12), and the mean ± SD was plotted for each time point. Levels of fluorescence were normalized to the value just after the metaphase alignment. Arrowheads indicate the time points when cyclin B1 begin to degrade. (C) Quantification of the cyclin B1 degradation rate. The degradation rate of cyclin B1 intensities from 12 cells were averaged for each experiment and are shown in a histogram with SEM. A.U., arbitrary unit. Bar, 10 µm.

Figure 5.

The kinetochore stretching is essential to silence the SAC. (A) A model illustrating the kinetochore stretching and the SAC silencing. Biorientation induces stretching of centromeres (modeled as a spring). Normally the kinetochores (yellow amorphous structures) undergo stretching, as denoted by arrows, and this kinetochore stretching promotes SAC inactivation. If kinetochore stretching is inhibited (e.g., by low nocodazole treatment or by condensin I depletion), SAC is maintained. (B) Proximal and distal kinetochores of monooriented chromosomes (top) were assessed for microtubule attachments by Mad2 recruitment as in Fig. 2 A (bottom). Monooriented chromosomes are indicated by arrowheads. (C) Stretching is induced at proximal but not at distal kinetochores of monooriented chromosomes. Profiles of the intra- and interkinetochore lengths and distances were assessed with the threshold values defined in Fig. 1 B (red dotted lines). The incidences for the kinetochore stretching were 13.2% and 0.0%, and the interkinetochore distances were 0.51 ± 0.02 µm (mean ± SEM) and 0.54 ± 0.02 µm for proximal (n = 38, 24 cells) and distal (n = 39, 24 cells) kinetochores, respectively. Bar, 5 µm.