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. 2009 Jul 28;19(14):1176-81.
doi: 10.1016/j.cub.2009.05.060. Epub 2009 Jul 9.

A novel protein phosphatase 1-dependent spindle checkpoint silencing mechanism

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A novel protein phosphatase 1-dependent spindle checkpoint silencing mechanism

Vincent Vanoosthuyse et al. Curr Biol. .

Abstract

The spindle checkpoint is a surveillance system acting in mitosis to delay anaphase onset until all chromosomes are properly attached to the mitotic spindle. When the checkpoint is activated, the Mad2 and Mad3 proteins directly bind and inhibit Cdc20, which is an essential activator of an E3 ubiquitin ligase known as the anaphase-promoting complex (APC). When the checkpoint is satisfied, Cdc20-APC is activated and polyubiquitinates securin and cyclin, leading to the dissolution of sister chromatid cohesion and mitotic progression. Several protein kinases play critical roles in spindle checkpoint signaling, but the mechanism (or mechanisms) by which they inhibit mitotic progression remains unclear. Furthermore, it is not known whether their activity needs to be reversed by protein phosphatases before anaphase onset can occur. Here we employ fission yeast to show that Aurora (Ark1) kinase activity is directly required to maintain spindle checkpoint arrest, even in the presence of many unattached kinetochores. Upon Ark1 inhibition, checkpoint complexes are disassembled and cyclin B is rapidly degraded. Importantly, checkpoint silencing and cyclin B degradation require the kinetochore-localized isoform of protein phosphatase 1 (PP1(Dis2)). We propose that PP1(Dis2)-mediated dephosphorylation of checkpoint components forms a novel spindle checkpoint silencing mechanism.

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Figures

Figure 1
Figure 1
Ark1 Activity Is Required to Maintain the Mitotic Checkpoint Complex/Anaphase-Promoting Complex and Cell-Cycle Arrest (A) In the presence of the ATP analog 1NMPP1, Ark1 is the only kinase directly inhibited in ark1-as3 cells . The spindle checkpoint was activated via the cold-sensitive tubulin mutant nda3-KM311. After 6 hr at the restrictive temperature, 80%–90% of cells were arrested with high levels of cyclin B on spindle pole bodies (SPBs), and all kinetochores were unattached. At this point, DMSO (control) or 1NMPP1 was added to the culture to inhibit Ark1, and the levels of cyclin B were monitored by live-cell imaging. In the presence of 1NMPP1, cyclin B levels dropped rapidly, demonstrating that Ark1 activity is required to maintain spindle checkpoint arrest in response to unattached kinetochores. The time after DMSO or 1NMPP1 addition is indicated in minutes. (B) Cells were fixed 40 min after addition of 1NMPP1. Most cells escaped arrest and underwent cytokinesis (arrows). (C) Top: after addition of 1NMPP1, a few cells remain arrested with high levels of cyclin B on SPBs (cell 1), whereas most escaped the arrest and precociously degraded cyclin B (cell 2). Bottom: the number of “escaped” cells was quantified in a 60 min time course. Data are derived from four independent experiments; error bars represent standard deviation. (D) 1NMPP1 addition and Ark1 inhibition have no effect on microtubule depolymerization in the nda3KM311 mutant. Anti-tubulin immunofluorescence confirms that at 18°C, all microtubules are depolymerized, whereas spindles are formed 15 min after release at 32°C. (E) Mitotic checkpoint complex/anaphase-promoting complex (MCC-APC) complexes are disassembled following Aurora inhibition with 1NMPP1. Lid1-TAP ark1-as3 Mad2-GFP Mad3-GFP nda3KM311 cells were arrested in mitosis (6 hr at 18°C), and the culture was divided before DMSO or 5 μM 1NMPP1 addition. Lid1p was immunoprecipitated from samples collected every 10 min and immunoblotted for associated Mad2-GFP and Mad3-GFP. (F) Model of MCC-APC binding.
Figure 2
Figure 2
PP1Dis2 Phosphatase Is Required for Checkpoint Silencing Following Ark1 Inhibition, and dis2 Mutants Have a Delay in Anaphase Onset upon Spindle Reassembly (A) When shifted to their restrictive temperature of 18°C, nda3-KM311 cells depolymerize microtubules and activate the spindle checkpoint with unattached kinetochores. Over time, nda3-KM311 and nda3KM311 dis2Δ cells accumulate in early mitosis with high levels of cyclin B (Cdc13-GFP) in the nucleus and on SPBs, showing that, unlike Mad2p, Dis2p is not required for checkpoint arrest. (B) The silencing assay was used to test whether phosphatases played a role in checkpoint silencing. Strikingly, when 1NMPP1 was added to the cultures, APC activation and cyclin B degradation were negligible in the absence of PP1Dis2 (dis2Δ), suggesting that PP1Dis2 is critical for checkpoint silencing upon Ark1 inhibition. This is a specific defect, because sds21Δ and clp1Δ mutants behaved like wild-type (as did PP2APar1 mutants; data not shown). For simplicity, the DMSO controls are not shown here. Data are derived from a minimum of three experiments; error bars represent standard deviation. (C and D) nda3KM311-arrested cells (6 hr at 18°C) were allowed to reform their spindle at the permissive temperature of 32°C (see Experimental Procedures). Progression through mitosis was then followed at regular intervals using spindle length as a marker, scoring the percentage of metaphase (C) and anaphase (D) cells. Upon recovery from nda3KM311 arrest, lack of Dis2p induced ∼20 min metaphase delay, whereas lack of Sds21p had no effect on the kinetics of anaphase onset.
Figure 3
Figure 3
Silencing Model Top: at unattached kinetochores, centromeric chromatin is not under tension, and centromere-localized Ark1 kinase (the green cloud of activity) is in close proximity to kinetochore-localized checkpoint components and can phosphorylate them efficiently, leading to effective APC inhibition. Bottom: stable attachments are formed after biorientation and lead to inter- and intrakinetochore stretching. This displaces Ark1 from kinetochore substrates and reduces its ability to phosphorylate checkpoint components, which can in turn be dephosphorylated by PP1Dis2 (orange clouds of activity), leading to checkpoint silencing.

Comment in

  • Cell division: righting the check.
    Fuller BG, Stukenberg PT. Fuller BG, et al. Curr Biol. 2009 Jul 28;19(14):R550-3. doi: 10.1016/j.cub.2009.06.047. Curr Biol. 2009. PMID: 19640489 Free PMC article. Review.

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