Figure 5.
Figure 5. Efficient checkpoint arrest requires the interaction of CCNB1 and MAD1. (A and B) HeLa cells were transfected with Myc-CCNB1 and either empty vector negative control (–), full-length mCh-MAD1, N-terminal (A) or C-terminal (B) MAD1 truncations. MAD1 IPs were performed using mCh-antibodies and Western blotted for CCNB1 with Myc-antibodies. (C) A schematic of the MAD1 truncations summarizes the CCNB1 binding data. (D) mCh-MAD1ƒ or MAD1101–718 were expressed in HeLa CCNB1-GFP cells depleted of endogenous MAD1 for 72 h with an siRNA duplex targeting the 3′ UTR of the MAD1 mRNA (siMAD1). Cells were then arrested in mitosis for 4 h with 0.3 µM nocodazole and 20 µM MG132. Mean kinetochore fluorescence ± SEM of MAD1 and CCNB1 normalized to the respective signal in control cells are plotted in the graph (n ≥ 12 cells per condition, 15 kinetochores per cell in three independent experiments). DNA was stained with DAPI. (E) mCh-MAD1ƒ or MAD1101–718 were expressed in HeLa MPS1-GFP cells depleted of endogenous MAD1 for 72 h with an siRNA duplex targeting the 3′ UTR of the MAD1 mRNA. Cells were then arrested in mitosis for 4 h with 0.3 µM nocodazole and 20 µM MG132 and stained for nuclear pore complexes (NPC). Graphs show the mean MAD1 or MPS1 levels ± SEM at unattached kinetochores (15 kinetochores per cell) in normal prophase (n = 3 cells per condition) and nocodazole-arrested mitosis (n = 12 cells per condition). (F) HeLa Flp-in/TREx GFP-MAD1ƒ, GFP-MAD1241–718, and GFP-MAD1101–718 cells were depleted of endogenous MAD1 for 72 h. Expression of GFP-MAD1 transgenes was induced for the last 48 h, and the cells were then imaged over 10 h in the presence of 0.3 µM nocodazole. The proportion of cells remaining in mitosis over a 600-min time course is plotted. HeLa cells depleted of CENP-E or TPR were checkpoint challenged and imaged in the same way. (G) MPS1 promotes recruitment of MAD1 to unattached kinetochores (shown as the yellow circle). CDK1-CCNB1 localizes to unattached kinetochores through an interaction with MAD1, creating a positive feedback loop counteracted by the action of PP2A-B55 on MPS1. MAD1 promotes formation of the APC/C inhibitor, mitotic checkpoint complex (MCC). This creates a second positive feedback loop preventing CCNB1 destruction. Solid lines indicate direct interactions (recruitment, phosphorylation, or dephosphorylation), whereas dotted lines indicate multistep processes. Components drawn outside the yellow circle are active globally rather than acting locally at the unattached kinetochore.

Efficient checkpoint arrest requires the interaction of CCNB1 and MAD1. (A and B) HeLa cells were transfected with Myc-CCNB1 and either empty vector negative control (–), full-length mCh-MAD1, N-terminal (A) or C-terminal (B) MAD1 truncations. MAD1 IPs were performed using mCh-antibodies and Western blotted for CCNB1 with Myc-antibodies. (C) A schematic of the MAD1 truncations summarizes the CCNB1 binding data. (D) mCh-MAD1ƒ or MAD1101–718 were expressed in HeLa CCNB1-GFP cells depleted of endogenous MAD1 for 72 h with an siRNA duplex targeting the 3′ UTR of the MAD1 mRNA (siMAD1). Cells were then arrested in mitosis for 4 h with 0.3 µM nocodazole and 20 µM MG132. Mean kinetochore fluorescence ± SEM of MAD1 and CCNB1 normalized to the respective signal in control cells are plotted in the graph (n ≥ 12 cells per condition, 15 kinetochores per cell in three independent experiments). DNA was stained with DAPI. (E) mCh-MAD1ƒ or MAD1101–718 were expressed in HeLa MPS1-GFP cells depleted of endogenous MAD1 for 72 h with an siRNA duplex targeting the 3′ UTR of the MAD1 mRNA. Cells were then arrested in mitosis for 4 h with 0.3 µM nocodazole and 20 µM MG132 and stained for nuclear pore complexes (NPC). Graphs show the mean MAD1 or MPS1 levels ± SEM at unattached kinetochores (15 kinetochores per cell) in normal prophase (n = 3 cells per condition) and nocodazole-arrested mitosis (n = 12 cells per condition). (F) HeLa Flp-in/TREx GFP-MAD1ƒ, GFP-MAD1241–718, and GFP-MAD1101–718 cells were depleted of endogenous MAD1 for 72 h. Expression of GFP-MAD1 transgenes was induced for the last 48 h, and the cells were then imaged over 10 h in the presence of 0.3 µM nocodazole. The proportion of cells remaining in mitosis over a 600-min time course is plotted. HeLa cells depleted of CENP-E or TPR were checkpoint challenged and imaged in the same way. (G) MPS1 promotes recruitment of MAD1 to unattached kinetochores (shown as the yellow circle). CDK1-CCNB1 localizes to unattached kinetochores through an interaction with MAD1, creating a positive feedback loop counteracted by the action of PP2A-B55 on MPS1. MAD1 promotes formation of the APC/C inhibitor, mitotic checkpoint complex (MCC). This creates a second positive feedback loop preventing CCNB1 destruction. Solid lines indicate direct interactions (recruitment, phosphorylation, or dephosphorylation), whereas dotted lines indicate multistep processes. Components drawn outside the yellow circle are active globally rather than acting locally at the unattached kinetochore.

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