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A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells.

Wang XM, Zhai Y, Ferrell JE - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305-5332, USA.

ABSTRACT
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.

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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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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.

Mentions: Therefore, we chose to identify and microinject mitotic cells first, and then immediately thereafter treat them with nocodazole. We found that cells injected with wild-type XCL100 protein during prometaphase or metaphase gradually began to exit mitosis ∼45 min to 1 h after nocodazole treatment, without re-forming spindles or carrying out sister chromatid segregation, karyokinesis, or cytokinesis (Table I). Initially, the chromosomes became clustered and then decondensed, and finally the nuclear envelope reformed and nucleoli appeared (Fig. 5). The resulting cells resembled typical, flat interphase cells, except that the cells and their nuclei were unusually large (Fig. 5). When cells were injected during prophase (before nuclear envelope breakdown) and then treated with nocodazole, they did not exit mitosis.


A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells.

Wang XM, Zhai Y, Ferrell JE - J. Cell Biol. (1997)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139774&req=5

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.
Mentions: Therefore, we chose to identify and microinject mitotic cells first, and then immediately thereafter treat them with nocodazole. We found that cells injected with wild-type XCL100 protein during prometaphase or metaphase gradually began to exit mitosis ∼45 min to 1 h after nocodazole treatment, without re-forming spindles or carrying out sister chromatid segregation, karyokinesis, or cytokinesis (Table I). Initially, the chromosomes became clustered and then decondensed, and finally the nuclear envelope reformed and nucleoli appeared (Fig. 5). The resulting cells resembled typical, flat interphase cells, except that the cells and their nuclei were unusually large (Fig. 5). When cells were injected during prophase (before nuclear envelope breakdown) and then treated with nocodazole, they did not exit mitosis.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305-5332, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus