Quantitative measurement of the catastrophe rate of dynamic microtubules.
Previous work has shown that catastrophe frequency is the predominant dynamic parameter of microtubules that changes dramatically during the cell cycle. As an alternative to videomicroscopy assays, we have developed a biochemical 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-32P-GTP, followed by a cold GTP chase. The rate of loss of 32P label in microtubules measured by this method is equal to the catastrophe frequency at microtubule plus ends measured by videomicroscopy of individual microtubules. Addition of mitotic extract from Xenopus eggs increases the catastrophe rate of purified tubulin by almost 100-fold, while interphase extract alters the catastrophe rate by about 20-fold as compared to pure tubulin. Most of the catastrophe-promoting activities in both mitotic and interphase extracts is found 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 rates. It will be of particular interest to search for catastrophe factors associated with intracellular membranes or other insoluble components.
Zhou
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BB
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kirschner
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MW
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002453:Cell Cycle; D004305:Dose-Response Relationship, Drug; D005976:Glutaral / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology*; D013696:Temperature; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*; D014981:Xenopus / Q000196:embryology
D014404:Tubulin; D008274:Magnesium; D002118:Calcium; D005976:Glutaral