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The Journal of Cell Biology, Vol 106, 2047-2056, Copyright © 1988 by The Rockefeller University Press
ARTICLES |
WG Dunphy and JW Newport
Department of Biology, University of California, San Diego, La Jolla 92093.
During the conversion to the mitotic state, higher eukaryotic cells activate a cascade of reactions which result in the disintegration of the nuclear envelope, the condensation of the DNA into chromosomes, and the reorganization of the cytoskeleton. In Xenopus, the induction of the mitotic state appears to be under the control of a cytoplasmic factor(s) known as mitosis-promoting factor or MPF. We have developed a rapid and highly sensitive version of an in vitro assay for MPF. The assay uses reconstituted nuclei in interphase cytoplasm from activated Xenopus eggs. The MPF-induced conversion from interphase to mitosis is conveniently monitored by the visual observation of the loss of the nuclear envelope from the substrate nuclei. At near saturating concentrations of MPF, nuclear breakdown requires 20-30 min. Preincubation experiments have revealed that the action of MPF requires only a few minutes and that the disassembly process itself takes up the remainder of the incubation period. Using this cell-free system, we have investigated the observation that protein synthesis is required for the progression through each successive mitotic cycle in the developing Xenopus embryo. A simple explanation for this finding would be that MPF is degraded after each mitosis and then resynthesized before the next mitotic cycle. However, using in vitro reactivation experiments, we have found that MPF is present in a latent, inactive form during interphase. These results suggest that the block in the cell cycle induced by inhibitors of protein synthesis is due to the lack of production of an activator of MPF.
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