A Wee1 checkpoint inhibits anaphase onset.

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Cyclin-dependent kinase 1 (Cdk1) regulates key cell cycle transitions, including mitotic entry and the metaphase-to-anaphase transition.
• Wee1 kinase activity is known to inhibit Cdk1, preventing mitotic entry.

Purpose of the Study:

• To investigate the role of the budding yeast Wee1 kinase, Swe1, in regulating the metaphase-to-anaphase transition.
• To elucidate the mechanism by which Swe1 controls anaphase onset and its relationship with the anaphase-promoting complex/cyclosome (APC).

Main Methods:

• Genetic manipulation of SWE1 and MIH1 genes in budding yeast.
• Analysis of cell cycle progression and anaphase onset timing.
• In vitro and in vivo assays to measure APC activity.
• Investigating the role of Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A), in the Swe1-dependent morphogenesis checkpoint.
• Site-directed mutagenesis of Cdk1 phosphorylation sites on the APC.

Main Results:

• Deletion of SWE1 or its opposing phosphatase MIH1 altered the timing of anaphase onset.
• Activation of the Swe1-dependent morphogenesis checkpoint or Swe1 overexpression led to metaphase arrest with reduced APC activity.
• The morphogenesis checkpoint's function was dependent on Cdc55.
• Checkpoint defects in cdc55Δ mutants were rescued by mutating Cdk1 phosphorylation sites on the APC, identifying the APC as a checkpoint target.

Conclusions:

• Swe1 restrains the metaphase-to-anaphase transition by preventing Cdk1-mediated phosphorylation and activation of the APC(Cdc20).
• Stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) likely trigger anaphase onset.
• These findings reveal a novel regulatory mechanism for APC activation and anaphase initiation involving Swe1 and PP2A(Cdc55).