Microtubule assembly in clarifiedXenopus egg extracts

Abstract

Crude cytoplasmic extracts made from Xenopus eggs have proven to be uniquely useful in the studies of the mechanism of spindle microtubule assembly dynamics and chromosome movement during progression through the cell cycle. We examined microtubule dynamic instability in the Xenopus system using video-enhanced differential interference contrast microscopy (VE-DIC), which required high-speed centrifugation in order to clarify crude Xenopus extracts of refractile particles. Surprisingly, the resultant clarified, undiluted extracts exhibited virtually no microtubule catastrophe, even in the presence of high MPF (cyclin B/p34cdc2 kinase) activity and mitogen-activated protein (MAP) kinase activity, a down-stream kinase also implicated in regulating microtubule dynamics. Microtubule elongation occurred at plus ends, and interphase microtubules grew at 17-30 microns/min while metaphase [meiotic, myelin basic protein kinase activity which is diagnostic for cytostatic factor (CSF)-arrested] microtubules grew at about 10 microns/min. Plus-end shortening rates for both interphase and metaphase extracts were > 50 microns/min. Addition of okadaic acid, a protein phosphatase inhibitor known to activate MAP kinase activity and cause an increase in microtubule turnover in extracts made from sea urchin eggs, had no effect on microtubule catastrophe in either interphase or metaphase Xenopus extracts. In addition, the microtubules assembled in interphase extracts were less sensitive to dilution than those in metaphase. This study is the first to describe the dynamic instability of microtubules in Xenopus extracts without the addition of exogenous tubulins or other buffer contaminants.