Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The spindle checkpoint ensures accurate chromosome segregation by preventing anaphase onset until all chromosomes are properly attached to the mitotic spindle.
• The mitotic checkpoint complex (MCC) inhibits the anaphase-promoting complex/cyclosome (APC/C) via CDC20, delaying mitosis until proper spindle attachment.
• While phosphorylation's role in the spindle checkpoint is known, the precise mechanisms regulating its maintenance and inactivation remain unclear.

Purpose of the Study:

• To investigate the role of mitotic phosphatases in regulating the spindle checkpoint.
• To determine how phosphatase activity influences the stability of the mitotic checkpoint complex (MCC) and the activation of the anaphase-promoting complex/cyclosome (APC/C).

Main Methods:

• Spindle checkpoint-arrested cells were treated with various phosphatase inhibitors.
• The effects of inhibitors on MCC stability and APC/C activation were assessed.
• Levels of cyclin A, cyclin B, and Cdk1 substrate phosphorylation were analyzed.

Main Results:

• Treatment with calyculin A and okadaic acid (1 μM) induced MCC dissociation and premature APC/C activation in checkpoint-arrested cells.
• This resulted in the degradation of cyclin A and cyclin B.
• Despite cyclin B degradation, cells failed to exit mitosis, indicated by sustained Cdk1 substrate phosphorylation and chromosome condensation.

Conclusions:

• Phosphatases are essential for maintaining the integrity of the MCC during spindle checkpoint arrest.
• Inhibition of phosphatases leads to premature APC/C activation but does not fully resolve mitosis, highlighting a critical role for phosphatases beyond MCC stability.