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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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Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1

Jonathan A Robbins1, Frederick R Cross

  • 1The Rockefeller University, New York, NY 10065, USA.

Molecular Biology of the Cell
|January 22, 2010
PubMed

Insights

Cyclin-dependent kinase (CDK) phosphorylation of the anaphase-promoting complex/cyclosome (APC/CC)-Cdh1 is essential for cell cycle progression in yeast. Disrupting this phosphorylation causes cell cycle arrest and spindle defects.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The anaphase-promoting complex/cyclosome (APC/CC)-Cdh1 regulates the cell cycle by targeting mitotic proteins for degradation.
  • Inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase is proposed to prevent premature substrate degradation.

Purpose of the Study:

  • To investigate the essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae.
  • To determine the roles of Cdh1 phosphorylation sites and interaction motifs in cell cycle regulation.

Main Methods:

  • Endogenous gene replacement of CDH1 with a CDK-unphosphorylatable mutant (CDH1-m11).
  • Analysis of cell cycle progression, spindle morphology, and protein degradation in CDH1-m11 cells.
  • Genetic manipulation including blocking proteolysis and altering gene dosage.

Main Results:

  • CDH1-m11 cells arrest in the first cell cycle with replicated DNA, sustained polarized growth, and monopolar spindles.
  • Neither Cdh1 polo kinase sites nor polo interaction motifs are required for cell cycle progression.
  • Expression of undegradable mitotic cyclin restores isotropic growth and spindle pole body (SPB) separation.
  • A minority of cells arrest with short bipolar spindles due to failure in Cdc20 accumulation and cohesin cleavage.
  • Bipolar spindle assembly is sensitive to the gene dosage of the Cdh1 inhibitor ACM1.

Conclusions:

  • CDK phosphorylation of Cdh1 is essential for timely exit from mitosis and proper cell cycle progression in yeast.
  • Different spindle-regulatory pathways exhibit distinct sensitivities to Cdh1 activity.
  • ACM1 may buffer the essential CDK phosphorylation of Cdh1, highlighting its regulatory role.

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