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Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
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Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Published on: December 5, 2017

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WT1 interacts with MAD2 and regulates mitotic checkpoint function

Jayasha Shandilya1, Eneda Toska1, Derek J Richard2

  • 1Department of Biological Sciences, University at Buffalo, Cooke Hall, North Campus, Buffalo, New York 14260, USA.

Nature Communications
|September 19, 2014
PubMed

Insights

The Wilms tumor 1 (WT1) protein, a tumor suppressor, regulates the mitotic checkpoint by interacting with MAD2. Depleting WT1 causes chromosome segregation defects, highlighting its role in genomic stability.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Tumor suppressors maintain genomic integrity by regulating the mitotic checkpoint.
  • The mitotic checkpoint complex (MCC) ensures accurate chromosome segregation during cell division.

Purpose of the Study:

  • To investigate the novel role of the tumor suppressor WT1 in regulating the mitotic checkpoint.
  • To elucidate the mechanism by which WT1 influences MCC function and chromosomal stability.

Main Methods:

  • Immunofluorescence to assess WT1 and MAD2 colocalization during mitosis.
  • Co-immunoprecipitation to study WT1 interactions with MAD2 and MCC components.
  • RNA interference (RNAi) to deplete WT1 and analyze its effects on mitotic progression and chromosome segregation.
  • Western blotting to evaluate the degradation of SECURIN and CYCLIN B1.

Main Results:

  • WT1 directly interacts with MAD2, a key spindle assembly checkpoint protein.
  • WT1 colocalizes with MAD2 during mitosis and binds preferentially to the C-MAD2 conformer.
  • WT1 associates with the MCC, prolonging the inhibition of the anaphase-promoting complex/cyclosome (APC/C).
  • WT1 depletion results in accelerated SECURIN turnover, reduced anaphase lag time, and chromosome segregation errors.

Conclusions:

  • WT1 functions as a crucial regulator of the mitotic checkpoint.
  • WT1 contributes to chromosomal stability through its interaction with the MCC.
  • These findings reveal a new mechanism for tumor suppression involving mitotic checkpoint control by WT1.

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