Quantitative measurement of the catastrophe rate of dynamic microtubules

Previous work has shown that catastrophe frequency is the predominant dynam ic parameter of microtubules that changes dramatically during the cell cycl e. As an alternative to videomicroscopy assays, we have developed a biochem ical assay to measure directly the average catastrophe rate of a population of microtubules. In this assay, the growing plus end of the microtubules, polymerized off seeds, are labeled with a brief pulse of alpha-P-32-GTP, fo llowed by a cold GTP chase. The rate of loss of P-32 label in microtubules measured by this method is equal to the catastrophe frequency at microtubul e plus ends measured by videomicroscopy of individual microtubules, Additio n of mitotic extract from Xenopus eggs increases the catastrophe rate of pu rified tubulin by almost 100-fold, while interphase extract alters the cata strophe rate by about 20-fold as compared to pure tubulin. Most of the cata strophe-promoting activities in both mitotic and interphase extracts is fou nd in particulate fractions. High-speed centrifugation of extracts appears to eliminate the components required for increasing microtubule catastrophe , but does not eliminate the cell cycle difference in microtubule dynamics. This assay provides a new approach to quantitate microtubule catastrophe r ates. It will be of particular interest to search for catastrophe factors a ssociated with intracellular membranes or other insoluble components. Cell Motil. Cytoskeleton 43:43-51, 1999. (C) 1999 Wiley-Liss, Inc.