Assay of membrane motility in interphase and metaphase Xenopus extracts.

Publisher Summary This chapter describes the production and use of Xenopus extracts to study the cell cycle regulation of microtubule-based motility. Xenopus extracts are prepared by using a slight modification of the method described by Murray and Kirschner for preparing cycling extracts. The main change is the inclusion of cycloheximide, which arrests the extracts in interphase. It is also possible to make extracts that appear to be in an interphase-like state without using electrical activation, simply by omitting ethylene glycol bis ( β -aminoethyl ether) N , N ′-tetraacetic acid (EGTA) from the buffers. For all experiments in which the cell cycle status needs to be clearly defined, it is preferable to use eggs that have been activated either electrically or by treatment with calcium ionophores. Meiotic extracts prepared from metaphase-arrested eggs can be induced to enter interphase by adding 1/50th volume of 20 m M CaCl 2 , 100 m M KCl, 1 m M MgCl 2 and cycloheximide to 100 μ g/ml, followed by incubation at 23°C for 30–45 minutes. After addition of EGTA to 0.4 m M , the sample is transferred to ice. The morphology of nuclei incubated in an extract provides a rapid assay for cell cycle status.