Abstract
ABSTRACTCenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.
Highlights
During normal mitosis, faithful chromosome segregation is assured by the robustness of kinetochore attachment to the spindle microtubules (K–MT attachments) and the surveillance mechanism called the spindle assembly checkpoint (SAC), which delays mitotic progression if incorrect K–MT attachments are detected, giving the cell time to correct them
The functionality of the checkpoint in cenp-metaD mutant cells was tested by first looking if there was a mitotic arrest in the presence of colchicine, a microtubule depolymerizing agent. cenp-metaD mutant neuroblasts accumulated in M phase after 1 hour colchicine treatment, as the mitotic index increased by 2.46-fold, comparable to the 3-fold increase observed in wild type
While cenp-metaD mutant cells arrested in mitosis when treated with colchicine, the relatively high level of cells with Premature Sister Chromatid Separation (PSCS) revealed that these cells were not able to maintain a prolonged arrest when treated with spindle poisons
Summary
Faithful chromosome segregation is assured by the robustness of kinetochore attachment to the spindle microtubules (K–MT attachments) and the surveillance mechanism called the spindle assembly checkpoint (SAC), which delays mitotic progression if incorrect K–MT attachments are detected, giving the cell time to correct them. Several checkpoint proteins (including Mad, Mad, Bub, BubR1, Bub and Mps1) bind to unattached or inappropriately attached kinetochores. This recruitment to the kinetochore generates a ‘stop anaphase’ signal that diffuses into the cytosol. This signal is composed of the checkpoint proteins Mad, BubR1 and Bub bound to Cdc, a key co-factor of the APC/C necessary for its activation
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