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The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.
Figure 1. The specificity of antibody X15. Immunoblots of lysates from XTC cells (lane 1), G2-phase oocytes (lane 2), and M-phase oocytes (lane 3) were probed with X15. XTC cells exhibited two bands: a lower band that comigrated with nonphosphorylated Erk2, and an upper band that comigrated with both Erk1 and phosphorylated Erk2. The upper band was identified as Erk1, rather than phosphorylated Erk2, based on its cross-reactivities with other Erk1 and Erk2 antisera, and on the fact that it did not shift to a lower apparent molecular weight when lysates were treated with XCL100 (not shown).
Figure 2. Immunolocalization of MAP kinase at different mitotic stages. XTC cells were fixed, permeabilized, and subjected to triple label staining with DAPI, X15, and a β-tubulin antibody (AâF), or with DAPI, X15 preimmune serum, and the tubulin antibody (G).
Figure 3. Single label immunolocalization of MAP kinase. XTC cells were fixed, permeabilized, stained with DAPI (not shown) and X15 or X15 plus blocking peptide (35 μM), and examined by fluorescence and phase microscopy. The cell shown is in metaphase.
Figure 5. Effects of perturbing MAP kinase function on the spindle assembly checkpoint. Mitotic cells were microinjected with the inactive XCL100 C260S mutant (left), wild-type XCL100 (middle), or XCL100 plus MKK-1* (right), and then treated with nocodazole. The morphological consequences were assessed by time-lapse video microscopy. Four images are shown from each video series. Numbers in each panel denote the time (in h and min) after nocodazole treatment.
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