Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.
The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.
Département de Biologie du Développement, Institut Jacques Monod, CNRS-Université Paris VII, France.
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de Pennart
M H
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Verlhac
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C
Cibert
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A
Santa Maria
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B
Maro
D000818:Animals; D001345:Autoradiography; D002503:Centromere / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005260:Female; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D008677:Metaphase / Q000187:drug effects; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D019319:Okadaic Acid; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D013462:Sulfur Radioisotopes; D013997:Time Factors
D004988:Ethers, Cyclic; D008869:Microtubule-Associated Proteins; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D019319:Okadaic Acid; D008715:Methionine; D006656:Protamine Kinase; D010749:Phosphoprotein Phosphatases