Calpains form a superfamily of Ca2+-dependent intracellular cysteine proteases with various isoforms. Two isoforms, μ- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca2+ concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not μ-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.
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