ASSEMBLY IN CLARIFIED XENOPUS EGG EXTRACTS

Crude cytoplasmic extracts made from Xenopus eggs have proven to be un iquely useful in the studies of the mechanism of spindle microtubule a ssembly dynamics and chromosome movement during progression through th e cell cycle. We examined microtubule dynamic instability in the Xenop us system using video-enhanced differential interference contrast micr oscopy (VE-DIC), which required high-speed centrifugation in order to clarify crude Xenopus extracts of refractile particles. Suprisingly, t he resultant clarified, undiluted extracts exhibited virtually no micr otubule catastrophe, even in the presence of high MPF (cyclin B/p34(cd c2) kinase) activity and mitogen-activated protein (MAP) kinase activi ty, a downstream kinase also implicated in regulating microtubule dyna mics. Microtubule elongation occurred at plus ends, and interphase mic rotubules grew at 17-30 mu m/min while metaphase [meiotic, myelin basi c protein kinase activity which is diagnostic for cytostatic factor (C SF)-arrested] microtubules grew at about 10 mu m/min. Plus-end shorten ing rates for both interphase and metaphase extracts were >50 mu m/min . Addition of okadaic acid, a protein phosphatase inhibitor known to a ctivate MAP kinase activity and cause an increase in microtubule turno ver in extracts made from sea urchin eggs, had no effect on microtubul e catastrophe in either interphase or metaphase Xenopus extracts. in a ddition, the microtubules assembled in interphase extracts were less s ensitive to dilution than those in metaphase. This study is the first to describe the dynamic instability of microtubules in Xenopus extract s without the addition of exogenous tubulins or other buffer contamina nts. (C) 1997 Wiley-Liss, Inc.