Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest.
In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.
Tunquist
Brian J
BJ
Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, 4200 E. 9th Avenue, Campus Box C236, Denver, CO 80262, USA.
Eyers
Patrick A
PA
Chen
Lin G
LG
Lewellyn
Andrea L
AL
Maller
James L
JL
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018816:Genes, cdc / Q000187:drug effects / Q000502:physiology; D064246:Mad2 Proteins; D008540:Meiosis / Q000187:drug effects / Q000502:physiology*; D008677:Metaphase / Q000187:drug effects / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D017232:Oncogene Proteins v-mos / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009866:Oogenesis / Q000187:drug effects / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism* / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017231:Proto-Oncogene Proteins c-mos / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism* / Q000494:pharmacology; D014982:Xenopus laevis
D000906:Antibodies; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D017232:Oncogene Proteins v-mos; D010750:Phosphoproteins; D012097:Repressor Proteins; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D017231:Proto-Oncogene Proteins c-mos