Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase.
Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase.
Molecular Cancer Research, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands.
Timo E F
TE
Kuijt
Manja
M
Omerzu
Adrian T
AT
Saurin
Geert J P L
GJ
Kops
D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; D009687:Nuclear Proteins