CENP-E as an essential component of the mitotic checkpoint in vitro

Accurate chromatid separation is monitored by a checkpoint mechanism that d elays anaphase onset until all centromeres are correctly attached to the mi totic spindle. Using Xenopus egg extracts, the kinetochore-associated micro tubule motor protein CENP-E is now found to be required for establishing an d maintaining this checkpoint. When CENP-E function is disrupted by immunod epletion or antibody addition, extracts fail to arrest in response to spind le damage. Mitotic arrest can be restored by addition of high levels of sol uble MAD2, demonstrating that the absence of CENP-E eliminates kinetochore- dependent signaling but not the downstream steps in checkpoint signal trans duction. Because it directly binds both to spindle microtubules and to the kinetochore-associated checkpoint kinase BUBR1, CENP-E is a central compone nt in the vertebrate checkpoint that modulates signaling activity in a micr otubule-dependent manner.