The spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components.
- Received July 20, 2016.
- Accepted August 25, 2016.
- © 2016. Published by The Company of Biologists Ltd
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