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Tension-sensitive kinetochore phosphorylation in vitro

R B Nicklas1, M S Campbell, S C Ward

  • 1Department of Zoology, Duke University, Durham, NC 27708, USA. bnicklas@acpub.duke.edu

Journal of Cell Science
|October 9, 1998
PubMed
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Cellular checkpoints detect misattached chromosomes via tension-sensitive kinetochore phosphorylation. This study investigates the in vitro mechanism of this tension-mediated dephosphorylation, identifying key components.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Cell division relies on accurate chromosome segregation.
  • A checkpoint delays anaphase upon detecting misattached chromosomes, preventing errors.
  • Kinetochore protein phosphorylation is implicated in sensing chromosome attachment tension.

Purpose of the Study:

  • To investigate the in vitro mechanism of tension-mediated dephosphorylation at kinetochores.
  • To identify the tension-sensitive component in the kinetochore phosphorylation system.
  • To explore the evolutionary implications of chromosome checkpoint detection.

Main Methods:

  • Established an in vitro system using washed chromosomes from mammalian and insect cells.
  • Utilized ATP and phosphatase inhibitors to induce kinetochore phosphorylation.

Related Experiment Videos

  • Analyzed the effects of a mammalian mitotic kinase on insect meiotic chromosome phosphorylation.

    • Main Results:

    • Confirmed that kinetochores contain a complete phosphorylation system (kinase, phosphatase, substrate).
    • Demonstrated that in vitro phosphorylation is tension-sensitive, mirroring in vivo conditions.
    • Identified the kinase or substrate, not the phosphatase, as the tension-sensitive component.
    • Showed mammalian kinase under-phosphorylates insect X-chromosomes, similar to native insect kinase.

    Conclusions:

    • The in vitro system accurately reflects in vivo tension-sensitive kinetochore phosphorylation.
    • The study implicates either the kinase or substrate as the tension sensor.
    • Findings offer insights into the evolution of checkpoint-evading chromosomes and kinase-substrate accessibility.