p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment - PubMed (original) (raw)

p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment

Robert S Hagan et al. Mol Biol Cell. 2011 Nov.

Abstract

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a "wait anaphase" signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31(comet), a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31(comet) during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31(comet) traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31(comet) arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31(comet) is required for timely mitotic exit. We propose that p31(comet) is an essential component of the machinery that silences the checkpoint during each cell cycle.

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Figures

FIGURE 1:

p31comet and Mad2 act in opposition to promote anaphase onset. (A) p31comet overexpression and Mad2 depletion cause premature anaphase. HeLa Histone 2B-mRFP cells were transfected with GFP or GFP-p31comet, and HeLa Histone 2B-GFP cells were transfected with Mad2 siRNA or control siRNA and followed by time-lapse microscopy. The times represent minutes after NBD; scale bar, 10 μm. The graph shows the fraction of cells undergoing premature anaphase, defined as chromosome segregation at T < 18 min. (B) p31comet depletion and Mad2 overexpression arrest cells in mitosis. As in (A), HeLa Histone 2B-GFP cells were transfected with p31comet or control siRNA, and HeLa Histone 2B-mRFP cells were transfected with GFP-Mad2. The graph depicts a fraction of cells arrested in mitosis, defined as still in metaphase at T = 45 min. (C) Immunoblots of whole-cell lysates from HeLa cells treated with siRNA against lamin A (control), p31comet, or Mad2. Blots were probed with antibodies as indicated.

FIGURE 2:

Cell cycle arrest following p31comet depletion is not due to defective kinetochore-MT attachments or sustained kinetochore signaling. (A) Cells depleted of p31comet arrest in mitosis. HeLa cells were treated with control or p31comet siRNA and stained with DAPI (blue) and anti-cyclin B or anti-cyclin A (green). (B) Kinetochores in p31comet-depleted cells are under tension. HeLa EGFP-CENP-A cells were transfected with siRNAs against p31comet or a control, and interkinetochore distances were measured in metaphase cells using a kinetochore-tracking assay. Interkinetochore distances are represented as whisker plots based on 240 (p31comet) and 319 (control) sister-kinetochore pairs in two independent experiments. (C) Checkpoint proteins localize normally in p31comet-depleted cells. HeLa cells depleted for p31comet or Lamin A control were stained with DAPI (blue) and antibodies against the indicated checkpoint proteins (red and green). Scale bars, 10 μm.

FIGURE 3:

p31comet localizes to unattached kinetochores in a Mad2-dependent manner. (A) p31comet localizes to unattached kinetochores. HeLa cells were stained with DAPI, anti-p31comet antibodies, and CREST serum. Scale bar, 5 μm. (B) p31comet binds to kinetochores in prometaphase and requires Mad2. HeLa cells treated with the indicated siRNA were stained with DAPI, CREST, and anti-p31comet antiserum. Scale bar, 2.5 μm. Indicated sister kinetochore pairs are magnified at right; scale bar, 0.5 μm. (C) Quantitation of p31comet loss from kinetochores. p31comet/CREST signal ratios from individual kinetochores are plotted as dots; bars represent mean ratio values. (D) Images captured from a time-lapse sequence showing p31comet–EYFP localization during mitosis. Kinetochore localization (arrows) is most apparent in early prometaphase when chromosomes are unattached. Upon attachment a portion of the fluorescence accumulates at spindle poles (arrowheads) and in the spindle region (triangles). After all chromosomes no longer have kinetochore staining, anaphase onset begins (dashed lines mark chromosome masses). Time points are in minutes. Scale bar, 10 μm.

FIGURE 4:

p31comet acts downstream of the timer functions of Mad2 but upstream of kinetochore functions. (A) Mitotic arrest of HeLa Histone 2B-GFP cells was measured as in Figure 1B. Bars indicate mean and range of at least two independent experiments. (B and C) Cumulative frequency graphs of anaphase times in cells treated with siRNA as indicated. Dashed lines for control RNAi and p31comet/control RNAi are identical between B and C.

FIGURE 5:

Trafficking of p31comet on and off kinetochores. (A) An image sequence showing FRAP of p31comet–EYFP at an unattached kinetochore. The inset shows the bleached kinetochore recovering in detail. Time points are in seconds. Scale bar, 10 μm. (B) Recovery curve for p31comet–EYFP at unattached kinetochores in untreated PtK2 cells. (C) Recovery curve for p31comet–EYFP at unattached kinetochores in nocodazole-arrested PtK2 cells. (D) An image sequence showing FRAP of p31comet–EYFP in the nucleoplasm and nuclear envelope. Time points are in seconds. Scale bar, 5 μm. (E) Recovery curve for p31comet–EYFP at the nuclear envelope in PtK2 cells shown as a percentage of the original fluorescence intensity over time. A biphasic curve had the best fit to the data. The fast phase is the diffusible nucleoplasmic fraction of p31comet–EYFP, whereas the slow fraction is p31comet at the nuclear envelope.

FIGURE 6:

Computer-driven simulations demonstrating that p31comet levels can control APC/C activity. APC/C activation state as a function of active p31comet concentration in the presence (A) or absence (B) of kinetochore signaling reveals a dependence of p31comet on APC/C activation. (C) Checkpoint protein complex dynamics are shown for two concentrations of p31comet. The dashed line illustrates the addition of a low (50 nM) concentration of p31comet, and the solid line the addition of p31comet to a high level (100 nM). The high levels of p31comet result in a rapid 80% inhibition of the kinetochore pool (1 nM K-C-Mad2 present) but much lower (40%) in the lower p31comet simulation. Through the combined action of C-Mad2 binding and C-Mad2–APC dissociation, the APC/C is partitioned between two interconverting inactive pools of p31:C-Mad2:APC/C and C-Mad2:APC/C. The result is that the APC/C is rapidly activated at the high p31comet level, whereas it is inactive at the low level.

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p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment - PubMed (original) (raw)