Homeostatic Control of Mitotic Arrest Gianluca Varetti, Claudia Guida, Stefano Santaguida, Elena Chiroli, Andrea Musacchio  Molecular Cell  Volume 44, Issue 5, Pages 710-720 (December 2011) DOI: 10.1016/j.molcel.2011.11.014 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 p31comet Interacts with the APC/C and MCC in Mitosis (A) HeLa cells were either left untreated (asynchronous), arrested in nocodazole for 16 hr or released in fresh medium for 2 hr. S/G2 indicates adherent cells at the time of mitotic shake off. Lysate (1.3 mg) was used for immunoprecipitation with anti-p31comet antibody, followed by western blotting against the indicated proteins. (B) HeLa cells were treated with nocodazole (80 ng/ml) overnight. Mitotic cells were collected by shake off and the APC/C was immunopurified from 3 mg of lysates. The unbound fraction (indicated as SN) underwent an anti-Cdc20 immunoprecipitation. The indicated proteins were detected by western blot analysis. Myc IP: Immunoprecipitation with an anti-myc antibody was used as negative control. The two panels of each row were acquired from the same film and intervening lanes were removed. (C) HeLa FlpIn T-REX cells were transfected with a vector expressing wild-type or Q83A-F191A FLAG-HA-p31comet (QF). Transgene expression in the resulting stable lines was induced with 1 ng/ml doxycycline. Ten hours after induction, cells were treated with nocodazole for 15 hr. Mitotic cells were collected and immunoprecipitation with an anti-FLAG antibody was carried out with 1.5 mg of lysate and proteins were detected by western blotting. See also Figures S1 and S2. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Requirement of p31comet for Mitotic Degradation of Cdc20 and MCC Disassembly (A) HeLa cells synchronized through subsequent thymidine-aphidicolin blocks (abbreviated as TAB) were transfected twice with control siRNAs or siRNAs for p31comet. Cells were then treated with nocodazole for 7 hr. Mitotic cells were collected by shake off and CHX added at 20 μg/ml. Cells were harvested at the indicated times after CHX addition and analyzed by western blotting. The asterisk marks an unspecific band recognized by the anti-Cdc20 antibody. (B) HeLa cells were synchronized with a thymidine-aphidicolin block and transfected twice with a control siRNA or siRNAs for p31comet. Cells were blocked in 80 ng/ml nocodazole and then treated with 10 μM MG132 for the indicated times. Mitotic cells were collected by shake off and Cdc20 was immunoprecipitated from 0.7 mg of lysates. Western blotting was performed to detect Cdc20. (C) HeLa cells were treated as in (A) and blocked in nocodazole. Ten micromolar MG132 was added for 2 hr where indicated. The APC/C was immunoprecipitated and western blotting performed to detect the indicated proteins. (D) Quantification of the immunoprecipitated proteins was performed by densitometric analysis of five experiments conducted as in (C). Data are represented as mean ± SEM. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 p31comet Is Required for Checkpoint Adaptation in a Prolonged Mitotic Arrest (A) HeLa cells were interfered as in Figure 2A. Cells were then treated with nocodazole. After 7 hr, mitotic cells were collected and the APC/C was immunopurified. Ub ligase activity was assayed in vitro on Cyclin B11–87. (B) HeLa cells were treated as in (A). After 7 hr in nocodazole, mitotic cells were collected and imaged. The relative cumulative frequency of mitotic exit times (marked by cell flattening) is displayed. A whiskers and box graph of the same populations (showing median, 25th and 75th percentile, and minimum and maximum values) is also shown. Number of cells (n): control RNAi = 58; p31comet RNAi = 67. For the p31comet RNAi condition, median, 25th and 75th percentile, and maximum have the same value, since most of the cells remained mitotic until the end of the experiment. (C) HeLa cells were interfered as in Figure 2A, and directly released in nocodazole (80 ng/ml). After 12 hr, mitotic cells were collected through shake off and plated in different dishes in the continuous presence of nocodazole. Cells were harvested at the indicated times after the addition of nocodazole and analyzed by western blotting. S/G2 indicates the cells that were still adherent at the moment of mitotic shake off. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Protein Synthesis in Mitosis Is Required for MCC Formation and Robust SAC Response (A) HeLa cells were treated with CHX as in Figure 2A. After 90 min, mitotic cells were collected, the APC/C was immunopurified and analyzed by western blotting. (B) Protein extracts from cells treated as in (A) underwent western blotting to detect the indicated proteins. Quantification from three experiments is reported in Figure S3. (C) HeLa cells were interfered as in Figure 2A and treated with nocodazole. After 4 hr, CHX was added where indicated and cells were imaged. A cumulative frequency graph of the timing of mitotic exit is shown. (D) A whiskers and box graph of the experiment in (C) is shown. n: control RNAi = 46; control RNAi + CHX = 37; p31comet RNAi = 38; p31comet RNAi + CHX = 43. (E) HeLa cells were treated as in (A). CHX was left for 110 min where present. APC/C immunopurified from mitotic lysates was assayed in vitro toward Cyclin B11–87. –APC: the assay was performed by mock-precipitating the APC/C from untreated control cells. (F) HeLa cells were treated with nocodazole for 12 hr. Mitotic cells were collected by shake off, replated, and CHX was added at 20 μg/ml. Cells were harvested at the indicated times after CHX addition and analyzed by western blotting. Serial dilutions of a lysate of untreated mitotic cells were loaded to compare the relative levels of Cdc20. See also Figure S3. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 p31comet-Mediated MCC Turnover in Prometaphase Is Required for Timely Mitotic Exit (A) HeLa cells were interfered as in Figure 2A and then treated with nocodazole. After 7 hr, mitotic cells were collected or released in fresh medium for 1 or 2 hr. The APC/C was immunopurified and analyzed by western blotting. (B) The APC/C was immunopurified from noccodazole-released cells as in (A). Ub ligase activity was assayed in vitro on Cyclin B11–87. (C) HeLa cells were treated as in (A) and released in nocodazole. MG132 was added where indicated and left 2 hr. Mitotic cells were released in fresh medium and imaged. A cumulative frequency graph of the timing of mitotic exit is shown. A whiskers and box graph displaying the smallest observation (sample minimum), lower quartile (Q1), median (Q2), upper quartile (Q3), and largest observation (sample maximum) of the same populations is shown. n: control RNAi = 83; control RNAi + MG132 = 101; p31comet RNAi = 79; p31comet RNAi + CHX = 70. For the p31comet RNAi condition, the 75th percentile and maximum have the same value. Two-tailed p value between the times of mitotic exit relative to control and MG132-treated populations is < 0.0001, calculated through a Mann-Whitney test. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 p31comet Is Required for Timely Anaphase Onset, but Not for Chromosome Congression, in Unperturbed Mitoses (A) HeLa cells stably expressing H2B-GFP were synchronized through a double thymidine block and transfected with control siRNAs or p31comet siRNAi (oligo 1). After release from the second arrest, cells were filmed. The time of completion of metaphase was determined with respect to the time of nuclear envelope breakdown (NEBD). The graph reports the relative cumulative frequency observed in the analyzed populations. n: control RNAi = 96 cells; p31comet RNAi = 60 cells. (B) The time of anaphase onset was determined with respect to the time of nuclear envelope breakdown (NEBD) and is shown in a whiskers and box graph recapitulating the smallest observation (sample minimum), lower quartile (Q1), median (Q2), upper quartile (Q3), and largest observation (sample maximum). n: control RNAi = 95 cells; p31comet RNAi, oligo 1 = 76 cells; p31comet RNAi, oligo 2 = 95 cells; p31comet RNAi, oligo 3 = 70 cells. (C) Still images from two representative cells utilized in the experiments in (A) and (B) and showing H2B-GFP fluorescence. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Homeostatic Control of Mitotic Arrest Homeostatic control of prometaphase. In prometaphase, unattached kinetochores promote the association of Mad2, BubR1:Bub3, and Cdc20 (which is under continuous synthesis) to generate MCC, which then binds to apo APC/C (APC/C) to generate the MCC:APC/C complex. p31comet associates with C-Mad2 in the MCC (APC/C:MCC:p31comet). With the contribution of E2 conjugating enzymes UbcH10 and Ube2S (Garnett et al., 2009; Williamson et al., 2009), this leads to polyubiquitylation of Cdc20 (APC/C:MCC-Ub:p31comet) and its destruction by the proteasome. Drawing of the APC/C is freely inspired by work on its structure (Herzog et al., 2009). See also Figure S4. Molecular Cell 2011 44, 710-720DOI: (10.1016/j.molcel.2011.11.014) Copyright © 2011 Elsevier Inc. Terms and Conditions