This chapter presents detailed methods for the preparation of cytostatic factor (CSF) extracts and for performing spindle assembly reactions. It also describes methods for depleting specific components from extracts, an approach that has been used successfully to determine the contributions of both motor and nonmotor components to spindle assembly. It also describes methods for analyzing anaphase in vitro. Mature Xenopus eggs are arrested in metaphase of meiosis II by CSF, which is thought to be the product of the c-mos protooncogene. Sperm entry triggers a calcium spike that initiates a series of events leading to the destruction of CSF and exit from the meiosis II metaphase arrest. This calcium sensitivity of the CSF arrest is exploited in the preparation of extracts by use of the calcium chelator EGTA. The presence of EGTA in buffers results in extracts that maintain the CSF arrest but can be induced to exit the CSF arrest by addition of calcium. This convenient control of cell cycle state allows one to easily obtain in vitro spindles with replicated chromosomes.
ASJC Scopus subject areas
- Cell Biology