Okadaic Acid Induces Interphase to Mitotic-like Microtubule Dynamic Instability by Inactivating Rescue

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 #M okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (<5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylationdependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening. T HE conversion from the long, persistent microtubules of interphase into the short, dynamic microtubules of mitosis is controlled by phosphorylation initiated by the activation of maturation promoting factor (MPF) (p34 ~ kinase) (4, 22, 45) and a cascade of downstream kinases, including MAP kinase (15, 16). Addition of either p34 ~2 (22, 45) or MAP kinase (16) to interphase cytoplasm or cytoplasmic extracts of Xenopus embryos produces conversion to short dynamic microtubules. Conversely, addition of drugs like 6-dimethylaminopurine, an inhibitor of p34 ~c2 kinase, induces long microtubules when added to mitotic cells (30). In both interphase and mitotic animal cells, the plus ends of microtubules exhibit dynamic instability, abruptly switching between persistent, constant velocity phases of elongation and shortening (7, 17, 34, 35). Microtubule dynamics and lengths depend on the switching frequencies, catastrophe and rescue, as well as on the velocities of elongation and shortening. Catastrophe and rescue frequencies are difficult to measure accurately because of their stochastic nature, and data must be obtained from measurements of individual microtubules (37, 47). There is now some information on how the parameters of microtubule dynamic instability change between interphase and mitosis (7, 17), and upon activation of p34 c~2 kinase in interphase cytoplasm with cyclin B (4). Okadaic acid inhibits the activity of the serine, threonine protein phosphatases 1 and 2a (PP1 and PP2a) ~ 1. Abbreviations used in this paper: PP1 and PP2a, protein phosphatases 1 and 2a. (5, 9, 18), and recent studies have shown that treatment with okadaic acid promotes premature and prolonged mitosis in sea urchin eggs (28) and shortens microtubules in interphase tissue culture cells (44). In the experiments reported here, we measured the changes in microtubule dynamics in interphase extracts of sea urchin eggs when phosphorylation was enhanced by inhibiting PP1 and PP2a with okadaic acid. Materials and Methods Preparation of Extracts Interphase extracts from Lytechinuspictus (Marinus, Long Beach, CA) eggs were made as previously described (Gliksman, N. R., and E. D. Salmon. 1987. Z Cell BioL 109:30a), from unfertilized eggs, or eggs 15 rain after fertilization. Gametes were isolated by intracoelomic injection of 0.56 M KCi. Sperm were collected dry and stored on ice for up to 5 h. Eggs were collected into and washed 3x with artificial sea water (Instant Ocean Aquarium Systems, Mentor, OH). Eggs were then dejellied by washing 2x in isotonic 19:1 (28 mM NaCI, 0.53 mM KC1, 5 mM Tris base, pH 8.0). Egg fertilization was carded out in sea water containing 3-amino-l,2,4triazole (1 mM) and passed through a l l0-#m mesh (Tetko Inc., Elmsford, NY) to remove the fertilization envelopes (36). Unfertilized eggs or eggs 15 rain after fertilization were collected in isotonic HEMG buffer (180 mM Hepes, pH 7.35, 10 mM EGTA, 10 mM MgCI2, 0.5 M glycine, 1 mM ATE 1 mM GTP, 1 mM DTT, and protease inhibitors: 0.3 mM phenyl methyl sulfonyl fluoride, 10 #g/ml p-toluene-sulfonyl-L-arginine methyl ester HC1, 40 #g/ml soybean trypsin inhibitor, 1 /~g/ml pepstatin, 1 #g/ml benzamidine HCI, 10 #g/ml aprotinin, and 1 #g/ml leupeptin) and packed with a minimum volume of buffer (20-30% dilution of the cytosol) using a hand centrifuge. The eggs were homogenized and centrifuged at 50,000 g for 45 rain at 3~ The clear middle layer was collected with a 25-gauge needle/syringe and used fresh or frozen in liquid nitrogen and stored at -80~ L. pictus egg extracts contained 15-19/~M tubulin (Western blot 9 The Rockefeller University Press, 0021-9525/92/12/1271/6 $2.00 The Journal of Cell Biology, Volume 119, Number 5, December 1992 1271-1276 1271 on May 3, 2019 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.119. 5.1271 Published Online: 1 December, 1992 | Supp Info: