Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts.

Accurate sister chromatid segregation is pivotal in the faithful transmission of genetic information during each cell division. To ensure accurate segregation, eukaryotic organisms have evolved a "mitotic (or spindle assembly) checkpoint" to prevent premature advance to anaphase before successful attachment of every chromosome to the microtubules of the mitotic spindle. An unattached kinetochore generates a diffusible signal that inhibits ubiquitination of substrates such as cyclin B and securins. This protocol presents an in vitro assay for studying the mitotic checkpoint using Xenopus laevis egg extracts. Meiotic spindles assembled around nuclei added to egg extracts are synchronized at metaphase by an endogenous activity known as cytostatic factor (CSF). Normally, the mitotic checkpoint results in continued metaphase arrest following inactivation of CSF activity (by addition of calcium) and disassembly of spindle microtubules (with a microtubule inhibitor such as nocodazole). Simple DAPI staining for chromatin structure or biochemical analysis of Cdc2/cyclin B (cyclin-dependent kinase) histone H1 kinase activity can be used to evaluate the stages of the cell cycle and the status of the mitotic checkpoint. This cell-free system derived from Xenopus eggs has been successfully used to unravel the mechanisms of mitosis, and it provides a distinct advantage over cell-based studies in which perturbing kinetochore functions often results in lethality.