Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55 - PubMed (original) (raw)
Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55
Claudio Vernieri et al. J Cell Biol. 2013.
Abstract
The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2A(Cdc55) dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2A(Cdc55) and APC/C(Cdc20) (i.e., a positive feedback loop) that controls APC/C(Cdc20) activity. The circuit could guarantee sustained APC/C(Cdc20) activity after Clb2 starts to be degraded.
Figures
Figure 1.
Cdh1 is dispensable for the adaptation to the spindle checkpoint. (A) Cdc28/Clbs trigger a chain of events that both promote (green) and inhibit (red) transition to anaphase. (B–D) GAL1-MAD2 (3X) (yAC489) and GAL1-MAD2 (3X) cdc27-5A cdc16-6A (yAC1675) cells were synchronized in G1 with α-factor and released in galactose-containing medium. 2 h after the release, α-factor was re-added. Samples were taken for Western blotting (B and D) and IF analysis (IF; C) with anti-tubulin antibodies. The data shown in C are from a single representative experiment out of three repeats. For the experiment shown, n = 100. (E) GAL1-MAD2 (3X) (yAC489), GAL1-MAD2 (3X) cdh1Δ (yAC1738), and GAL1-MAD2 (3X) cdc27-5A cdc16-6A (yAC1675) cells were grown at 30°C in YEPR and shifted to galactose. Samples were taken every hour and the proportion of metaphase spindles was measured by IF. The data shown are from a single representative experiment out of three repeats (n = 100).
Figure 2.
Cdc28 inhibition causes a decrease of Cdc20 levels during a checkpoint arrest. (A–D) CDC28 PDS1-MYC18 (yAC359) and cdc28-as1 PDS1-MYC18 (yAC779) cells were synchronized in G1 phase with α-factor and then released in YEPD supplemented with nocodazole. After 3 h in nocodazole, the indicated concentrations of 1-NMPP1 or DMSO were added together with 20 µg/ml of α-factor. Samples were taken at the indicated times after the addition of 1-NMPP1 or DMSO for Western blotting. (E, top) cdc28-as1 MAD2-3GFP NUF2-mCherry (yAC2182) cells were treated similarly to A–D: after 2.5 h in nocodazole (noco), cells were kept two additional hours in the presence of nocodazole supplemented with DMSO, 500 nM, or 5 µM 1-NMPP1 (120 min). One fraction of cells was instead released from nocodazole (Release). Colocalization of Mad2 with Nuf2 was scored at 120 min (see also Materials and methods); error bars refer to standard errors from three independent experiments. In each experiment, 50 cells were analyzed for each condition. (E, bottom) Examples of localized and nonlocalized Mad2.
Figure 3.
Cdh1 is responsible for Pds1 and Clb2 degradation upon strong Cdc28 inhibition. (A and B) GAL-SIC1 (yAC2025) and GAL-SIC1 cdh1Δ (yAC2023) cells were grown in either raffinose (A) or glucose (B) at 23°C. When in log phase, nocodazole was added to the media for 2.5 h and then cells were either supplemented with galactose (A) or kept in glucose (B). Samples were analyzed by Western blotting (top) and FACS (bottom). The data are from a single representative experiment out of three repeats.
Figure 4.
A minimal amount of 1-NMPP1 significantly delays adaptation to the spindle checkpoint. (A and B) GAL1-MAD2 (3X) cdc28as-1 PDS1-MYC18 (yAC1788) cells were grown in YEPR, synchronized in G1 with α-factor, and released in galactose. 1.5 h after the release, α-factor was re-added together with either DMSO or 50 nM of 1-NMPP1. Samples were treated for Western blotting and FACS (A), and IF analysis (B). The data shown are from representative experiments out of three repeats (in B, n = 100).
Figure 5.
Single cell analysis of adapting cells. (A) CLB2-GFP TUB2-Cherry GAL1-MAD2 (3X) (yAC1732) cells were grown at 30°C in synthetic medium containing raffinose, and then were either shifted to galactose (adapting cells; top) or left in raffinose (cycling cells; bottom). We show Clb2-GFP accumulation signal in the nucleus after smoothing (see Materials and methods). Individual traces are synchronized at the time when Clb2 starts increasing. (B) Top left, degradation rate of Clb2; top right, expression rate, i.e., the rate of Clb2 accumulation; bottom right, adaptation time; bottom left, maxima of Clb2 fluorescence. See also
Fig. S4
, Materials and methods, and
Video 1
. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 50. Cells were analyzed with the aid of the program phyloCell, written by G. Charvin (unpublished data).
Figure 6.
PP2ACdc55-mediated dephosphorylation of APC subunits is essential for spindle checkpoint activity. (A) CDC16-myc6 (yAC1936), CDC16-myc6 cdc55Δ (yAC1994), CDC27-myc9 (yAC1863), and CDC27-myc9 cdc55Δ (yAC1888) cells were grown in YEPD at 30°C, arrested in G1 phase, and released in nocodazole. Samples were taken during the G1 arrest and after 2 and 3 h for Western blotting analysis of Cdc16 (left) and Cdc27 (right) using Phos-tag reagent. The same samples were loaded on standard 10% acrylamide gels to assess the total levels of Pgk1, Cdc16, and Cdc27. In the bottom panels, the fraction of phospho-specific shifts on P-tag gels was normalized to the total amount of protein. Error bars refer to standard errors of three independent experiments. (B–E) GAL1-MAD2 (3X) PDS1-MYC18 cdc55Δ (yAC1823), GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A (yAC1675), and GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A cdc55Δ (yAC1952) cells were arrested in G1 with α-factor and released in galactose. Samples were collected for IF (B) and Western blotting analysis (C–E). The data shown in B are from a single representative experiment out of three repeats. For the experiment shown, n = 100.
Figure 7.
Cdc14 is neither sufficient nor necessary to bypass the spindle checkpoint. (A and B) net1Δ PDS1-MYC18 (yAC1999) and cdc55Δ PDS1-MYC18 (yAC1896) cells were grown at 25°C in YEPD, synchronized in G1 with α-factor, and released in the presence of nocodazole. 80 min after the release, when 90% of the cells were budded, α-factor was added again. Samples were collected for Western blotting and FACS. The data shown are from a single representative experiment out of three repeats. (C and D) GAL1-MAD2 (3X) PDS1-MYC18 (yAC489), GAL1-MAD2 (3X) PDS1-MYC18 cdc27-5A cdc16-6A (yAC1675), GAL1-MAD2 (3X) PDS1-MYC18 cdc14-1 (yAC1768), and GAL1-MAD2 (3X) PDS1-MYC18 cdc15-2 (yAC1780) cells were grown in YEPR at 23°C. They were synchronized in G1 with α-factor and released into galactose. After 2 h, the cultures were shifted to 37°C and resupplemented with α-factor. Samples were collected for Western blotting (C) and IF (D). The data shown in D are from single representative experiments out of three repeats. For the experiments shown, n = 100.
Figure 8.
A simple model for the metaphase-to-anaphase transition in adaptation. (A) APC/CCdc20, Cdc28/Clbs, and PP2ACdc55 give rise to a positive feedback loop (PFL) and a negative feedback loop (NFL). (B) The dynamics of the model are shown starting from the lowest Clb2 levels in both a regular transition to anaphase (solid line) and during adaptation (broken line; decreased activation rate of APC). See also Materials and methods.
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