Figure 1.
proTAME induces cohesion fatigue and prolonged mitotic arrest.
(A) Timeline showing the experimental strategy. HeLa cells were synchronised by a double thymidine block and then released into normal media for 4 hours before the addition of drugs. (B) Time-lapse sequences indicating the different phenotypes of cells treated with 10 µM proTAME. Numbers indicate the time from nuclear envelope breakdown (NEBD) in hours:minutes and the arrow points the appearance of cohesion fatigue. (C, D) Scatter plot showing the amount of time cells take from NEBD to mitotic exit after treatment with different concentrations of proTAME (C) or upon treatment with 15 µM proTAME, in combination with the Aurora B inhibitor ZM447439 or the Mps1 inhibitor AZ3146 (D). The horizontal line represents the mean and the colours represent the cell fate (see legend). At least 40 cells were analysed in each condition. P values were calculated using two-tailed Mann-Whitney test. (E) Model explaining how proTAME can cause a SAC-dependent mitotic arrest.
Figure 2.
proTAME induces SAC re-activation following loss of sister chromatid cohesion.
(A, B) Graphs measuring cyclin B fluorescence intensity upon proTAME treatment (5 µM), either in the absence (A) or presence (B) of AZ3146. In each case, T = 0 was normalised to the onset of metaphase or prometaphase respectively. Arrows indicate the time at which anaphase or cohesion fatigue occurs. Data from one representative cell is shown for each treatment.
Figure 3.
Increasing the level of cohesin on the chromosomes inhibits the mitotic arrest caused by proTAME.
(A) Immunoblot of HeLa cells treated showing Wapl depletion upon RNAi treatment. Bub3 was used as a loading control. (B) Immunofluorescence images of mitotic HeLa cells showing an increased amount of the cohesin subunit Smc3 on the chromosomes upon Wapl depletion. (C) Scatter plot of HeLa cells depleted of Wapl and treated with proTAME, showing an increase in the amount of time cells take to undergo cohesion fatigue. P values were calculated using two-tailed Mann-Whitney test. (D) Cell fate profiles of HeLa cells depleted of Wapl and treated with different concentrations of proTAME, as described in Fig. 1A. Each line represents an individual cell and different colours represent the different cell fates (see legend).
Figure 4.
Inhibition of cohesion fatigue promotes MCC disassembly, even when the proteasome is inhibited.
(A) Timeline showing the experimental strategy. (B) Immunoblots of BubR1 immunocomplexes isolated from HeLa cells. Cells were treated with Wapl RNAi, synchronised with a single thymidine block and then released into media containing nocodazole for 12 hours before being selectively detached and released into normal media or media containing MG132 for 3 hours. Cyclin B was used as a mitotic marker. (C) Bar graph quantifying the experiment in (B). Values represent the mean ± s.e.m. of three independent experiments, each analysed twice. P values were calculated using paired, two-tailed t-test.
Figure 5.
Proteasome activity is not required for MCC disassembly.
(A) Timeline showing the experimental strategy. (B) Immunoblot of BubR1 immune complexes isolated from HeLa cells arrested in mitosis with nocodazole and released into media containing different combinations of MG132, ZM447439 and AZ3146. Cyclin B and phosphorylated histone H3 Ser10 (pH3) were used as markers for mitosis and Aurora B activity respectively. (C) Bar graph quantifying the experiment in (B). Values represent the mean ± s.e.m. of three independent experiments.
Figure 6.
proTAME treatment does not prevent MCC turnover.
(A) Timeline showing the experimental strategy. (B, C) Immunoblots of BubR1 immune complexes isolated from mitotic HeLa cells arrested with nocodazole and released into media containing MG132, ZM447439 and AZ3146, in the presence or absence of 20 µM proTAME. Cyclin A was used as a marker for lack of APC/C activity. (C) Bar graph quantifying the experiment in (B). Values represent the mean ± s.e.m. of three independent experiments.
Figure 7.
APC/C activity is required for MCC disassembly independently of proteolysis.
(A) Timeline showing the experimental strategy. (B) Immunoblots of BubR1 immunocomplexes isolated from mitotic HeLa cells depleted of p31comet or Apc2 by RNAi, arrested in mitosis with nocodazole and released into media containing MG132, ZM447439 and AZ3146. Cyclin A was used as a marker for lack of APC/C activity. (C) Bar graph quantifying the experiment in (B). Values represent the mean ± s.e.m. of four independent experiments.
Figure 8.
APC/C activity is required for releasing itself from the MCC.
(A) Schematic representation of the strategy for the selective isolation of APC/C-MCC and free MCC. (B) Immunoblot of immunocomplexes isolated from mitotic HeLa cells released into media containing MG132, ZM447439 and AZ3146 after Apc2 RNAi. (C, D) Bar graph quantifying the amount of Mad2 bound to APC/C-MCC (C) or free-MCC (D) under the different conditions described in (A). Values represent the mean ± s.e.m. of three different experiments.