Research
Molecular Mechanism of the Spindle Checkpoint
The metaphase-to-anaphase transition occurs only after all chromosomes have attached properly to the mitotic spindle and aligned at the metaphase plate. Kinetochores that are not bound with spindle microtubules trigger the spindle checkpoint that inhibits the anaphase onset, thus ensuring accurate sister chromatid segregation during each cell division cycle. A defect in this process leads to aneuploidy that is associated with some genetic disorders, such as Down¡¦s syndrome, and cancers.
Our laboratory has been studied the molecular mechanism of the spindle checkpoint in budding yeast and the frog Xenopus laevis. We had systematically isolated homologues of the spindle checkpoint proteins in Xenopus, include Mad1, Mad2, Bub1, Bub3, and BubR1 (Mad3 in yeast). We have been studying their regulation and functional role using the cell-free system derived from the cytoplasm of frog eggs. This system provides the advantages of great cell cycle synchrony, the ability to reproduce cell cycle events in a test tube, and ease in manipulating its content. The spindle checkpoint involves protein kinases MAPK, Mps1, Bub1, and BubR1 and some of the spindle checkpoint components are phosphorylated during mitosis. We have demonstrated that MAPK phosphorylates Cdc20, Bub1, and Mps1, and that these phosphorylation events are important for the spindle checkpoint through different mechanisms. Phosphorylation of Cdc20 is required for Cdc20 to be bound with and inhibited by spindle checkpoint proteins, whereas Bub1 phosphorylation activates Bub1 at kinetochores and facilitates the spindle checkpoint in egg extracts. Phosphorylation of Mps1 by MAPK is required for kinetochore localization of Mps1 and other spindle checkpoint proteins. Together, these findings suggest that MAPK controls several major steps in the spindle checkpoint.
In yeast, we recently found that a mutant in the AAA ATPase Cdc48 cause metaphase arrest that is dependent on both the spindle assembly and spindle position checkpoints. It remains to be investigated how Cdc48 controls kinetochore-microtubule attachment and spindle position.
