TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

Abstract

Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

Keywords: Biological sciences; Cell biology; Molecular biology.

Graphical abstract

Figure 1

TORC1 inactivation overrides SAC-mediated metaphase arrest (A) Exponentially growing cells of strain SCU2755 (PDS1-3HA) were arrested in metaphase by nocodazole treatment for 3 h at 30°C. Thereafter, cells were treated with or without rapamycin for the indicated times. 3HA-tagged securin/Pds1 was detected by western blotting analysis using an anti-HA antibody. CDK was detected as a loading control. (B) Cells of strain SCU1964 (SCC1-TAP) were treated as described in (A). Cleaved products of Scc1 (shown by an arrow) were detected in a long-exposure image using an anti-TAP antibody (PAP). (C) Cells of strain SCU69 (CEN4-GFP), of which the vicinity of the centromere of chromosome IV was labeled with GFP, were arrested in metaphase as described in (A) and then incubated with or without rapamycin for 1 h. Images of cells, GFP and DAPI were captured using a microscope. Scale bar, 2.5 μm. Separated sister centromeres were counted and are expressed as percentages in Figure 2G, WT. (D) Cells of strain SCU2755 (PDS1-3HA) were arrested in metaphase and transferred to nitrogen-free media containing nocodazole. (E) Model of TORC inactivation-induced anaphase onset. Nutrient starvation sequentially causes the inactivation of TORC1, securin degradation, cohesin cleavage, and sister separation.

Figure 2

APC/C-Cdh1 mediates TORC1 inactivation-induced anaphase onset (A) Cells of strain SCU1712 (CLB5-TAP) were treated as described in Figure 1A. TAP-tagged cyclin B5 (Clb5) was detected by western blotting analysis using the anti-TAP antibody (PAP). (B–D) Cells of strains SCU1485 (BUB1-GFP) and SCU1337 (MAD2-GFP) harboring plasmid pSCU1701 (pMTW1-RFP) were treated as described in Figure 1C. Bub1-GFP and Mad2-GFP signals were captured with Mtw1-RFP (a kinetochore marker) signals. Scale bar, 2.5 μm. Percentages of cells with Bub1-GFP or Mad2-GFP on the kinetochore were determined, and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ns, not statistically significant. (E) Cells of strains SCU2755 (PDS1-3HA) and SCU2693 (PDS1-3HA cdc20-3) precultured at 25°C were arrested in metaphase by nocodazole treatment for 3 h and transferred to 37°C for 30 min. Thereafter, cells were treated with rapamycin (+Rap) or released into nocodazole-free media (-Noc) (Time 0). Cells of strain SCU2282 (PDS1-3HA cdh1Δ) were treated as described in Figure 1A. For comparison, see Figure 1A, WT. 3HA-tagged securin Pds1 was detected by western blotting analysis using the anti-HA antibody. (F) Cells of strains SCU69 (CEN4-GFP) and SCU2114 (CEN4-GFP cdh1Δ) were treated as described in Figure 1C. Scale bar, 2.5 μm. Percentages of cells with separated sister chromatids were determined and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p <0.05. (G) Five-fold serial dilutions of cells were spotted in 1-μL drops onto YPAD plates with or without 5 ng/mL rapamycin. The plates were incubated at 30°C for 1 day for YPAD plates and 2 days for rapamycin-containing YPAD plates. The yeast strains SCU893 (wild type) and SCU1228 (cdh1Δ) were used. (H) Model of APC/C-Cdh1 mediating TORC1 inactivation-induced anaphase onset. The inactivation of TORC1 sequentially causes APC/C-Cdh1 activation, degradation of securin and cyclin B5, and anaphase onset.

Figure 3

Cdc14 mediates TORC1 inactivation-induced anaphase onset (A) Cells of strain SCU3250 (PDS1-3HA cdc14-1) were treated as described in Figure 2E. 3HA-tagged securin Pds1 was detected by western blotting analysis using the anti-HA antibody. (B and C) Cells of strains SCU69 (CEN4-GFP) and SCU3204 (CEN4-GFP cdc14-1) preincubated at 25°C were arrested in metaphase by nocodazole treatment for 3 h and then transferred to 37°C for 30 min. Thereafter, the cells were further incubated with or without rapamycin for 1 h. Scale bar, 2.5 μm. Percentages of cells with separated sister chromatids are shown in (C). For the control, cells were observed 30 min after nocodazole removal. Averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p < 0.05; ∗∗; p < 0.0001. (D and E) Cells of strain SCU1000 (CDC14-5GFP) were treated as described in Figure 1C. Scale bar, 2.5 μm. Percentages of cells with diffused Cdc14 were determined and averages and SD from three independent experiments are shown in (E). For the control, cells were observed 30 min after nocodazole removal. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗∗∗∗, p <0.000001. (F) Five-fold serial dilutions of cells of strains SCU893 (wild type) and SCU1001 (cdc14-1) were spotted in 1-μL drops onto YPAD plates with or without 10 ng/mL rapamycin. The plates were incubated at 25°C for 3 days. An unrelated atg1Δ strain (SCU4067) defective in autophagy was used as the control. (G) Model of Cdc14 mediating TORC1 inactivation-induced anaphase onset. The inactivation of TORC1 sequentially causes Cdc14 activation, APC/C-Cdh1 activation, securin degradation, and anaphase onset.

Figure 4

TORC1 inactivation evokes Net1 degradation (A) Nocodazole-treated metaphase cells of strain SCU3260 (NET1-3HA) were treated with rapamycin or released into nocodazole-free media. 3HA-tagged Net1 was detected using the anti-HA antibody. (B) Cells of strain SCU7048 (NET1-3HA) arrested in metaphase by nocodazole treatment were transferred to nitrogen-free media containing nocodazole. Pgk1 was detected as a loading control. (C) Cells of the wild-type strain SCU893 harboring plasmid pSCU1819 (pGAL1-NET1-H6HAZZ) preincubated in a raffinose-based medium were supplemented with 0.01% galactose for 1 h for induction of NET1-H6HAZZ. Protein stabilization of Net1 was assessed after shutoff of NET1-H6HAZZ expression by glucose (2%) addition (time 0). Net1 levels were detected using the anti-ZZ antibody (PAP). (D) Cells of the wild-type strain SCU893 harboring pSCU1819 (pGAL1-NET1-H6HAZZ) preincubated in raffinose-based medium were arrested in metaphase by nocodazole treatment for 3 h. Thereafter, cells were supplemented with 0.01% galactose for 1 h for induction of NET1-H6HAZZ. Protein stabilization of Net1 was assessed in the presence or absence of rapamycin after shutoff of NET1-H6HAZZ expression by glucose addition (time 0). (E) Five-fold serial dilutions of cells were spotted in 1-μL drops onto YPAD plates with or without 5 ng/mL rapamycin. The plates were incubated at 30°C for 1 day for YPAD plates and 2 days for rapamycin-containing YPAD plates. The yeast strains SCU893 (wild type) and SCU1199 (CDC14TAB6-1) were used.

Figure 5

Net1 degradation elicits Cdc14 release in SAC-mediated metaphase cells (A) Five-fold serial dilutions of cells of strains SCU893 (NET1) and SCU3331 (net1-aid) were spotted in 1-μL drops onto YPAD plates with or without 1 mM 1-naphthaleneacetic acid (NAA). The plates were incubated at 30°C for 1 day. (B) Cells of strain SCU3331 (net1-aid) were treated with 1 mM NAA. Net1 levels were followed by western blotting analysis using an anti-IAA17 antibody. (C) Cells of strain SCU3331 (net1-aid) were arrested in metaphase by nocodazole treatment for 3 h and then Net1 degradation was induced by 1 mM NAA treatment (time 0). (D) Cells of strain SCU3409 (CDC14-5GFP net1-aid) were arrested in metaphase by nocodazole treatment for 3 h and then Net1 degradation was induced by 1 mM NAA treatment (time 0). Scale bar, 2.5 μm. Percentages of cells with diffused Cdc14 were determined and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗∗, p <0.0001. (E) Cells of strains SCU69 (CEN4-GFP) and SCU6232 (net1-aid CEN4-GFP) were arrested in metaphase by nocodazole treatment for 3 h at 30°C and then incubated with or without NAA for 1 h. Scale bar, 2.5 μm. Separated sister centromeres were counted and are expressed as percentages. Averages and SDs from three independent experiments are shown. Statistical analyses were carried out using the two-way ANOVA with Bonferroni correction. ∗, p <0.01.

Figure 6

TORC1 inactivation elicits mitotic slippage (A) Cells of strain SCU1709 (CLB2-TAP) were treated as described in Figure 1A. TAP-tagged cyclin B2 (Clb2) was detected by western blotting analysis using the anti-TAP antibody. (B and C) Cells of the wild-type strain SCU893 were arrested in metaphase by nocodazole treatment for 3 h and then further cultured with or without (control) rapamycin, or in nitrogen-depleted media (SD-N) (time 0) for the indicated times. Scale bar, 2.5 μm. Percentages of G1 cells without buds were determined and averages and SDs from three independent experiments are shown in (C). p Values were calculated using the two-way ANOVA with Bonferroni correction. ∗∗∗, p <0.0005. (D) Cells of wild-type strain SCU893 were cultured as described in (B) and subjected to flow cytometric analysis. Accumulation of G1 cells after rapamycin treatment or nitrogen starvation was assessed. (E) Model in which the inactivation of TORC1 elicits mitotic slippage. (F) The chromosome instability (CIN) assay using a tester strain SCU1237 was performed as described in STAR Methods.” Percentages of cells that lost their mini-chromosomes were determined, and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p <0.05. (G) Cells of strain SCU893 (wild type) were treated with rapamycin (200 ng/mL) and/or nocodazole (10 μg/mL) for 1 h. Thereafter, for detection of cell viability, cells were spread on the YPAD plate and incubated for 3 d at 30°C. The number of colonies formed from surviving cells was counted, and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p <0.01.

Figure 7

mTORC1 inactivation elicits mitotic slippage (A) A schematic of the experimental procedure. (B) Phase-contrast time-lapse imaging of nocodazole-treated A549 cells. An example representing each category of cell fate to classify cells in Figure S4A is shown. Numbers show elapsed time (hour:minute) relative to the start of imaging. Scale bar, 50 μm. (C) Fate of nocodazole-treated A549 cells with or without rapamycin treatment after 60-h imaging. Proportion of cells classified into each category, obtained by averaging the results of four independent experiments shown in Figure S4A, is presented. Error bars represent standard errors. (D) Rate of mitotic slippage in nocodazole-treated A549 cells with or without rapamycin treatment and/or Cdh1 depletion. Percentage of cells that underwent mitotic slippage among the cells that underwent mitotic cell death or mitotic slippage, obtained by averaging the results of four independent experiments that are shown as dots, is presented. Error bars represent standard errors. ∗∗, p <0.01; ∗∗∗, p <0.001 (Tukey's multiple comparison test). ns, not statistically significant. (E) Duration of mitosis in nocodazole-treated A549 cells that underwent mitotic cell death or mitotic slippage. The results are average of four independent experiments. The results of rare mitotic slippage events in cells depleted of Cdc20 with or without Cdh1 were not shown. Error bars represent standard errors. ∗∗∗, p <0.001 (Tukey's multiple comparison test, comparison with Mock -Rap, mitotic cell death). (F) Duration of interphase after mitotic slippage in nocodazole-treated A549 cells with or without rapamycin treatment and/or Cdh1 depletion. The results are average of four independent experiments, which are shown as dots. Error bars represent standard errors. ∗∗, p <0.01 (Tukey's multiple comparison test). ns, not statistically significant.