Tension-sensitive kinetochore phosphorylation and the chromosome distribution checkpoint in praying mantid spermatocytes

Abstract

Improper chromosome attachment to the spindle can lead to daughter cells with missing or extra chromosomes. Such mishaps are avoided in many cells by a checkpoint that detects even a single improperly attached chromosome. What is detected? A misattached chromosome is not under tension from opposed mitotic forces, and in praying mantid spermatocytes, direct experiments show that the absence of tension is what the checkpoint detects. How is the absence of tension detected? Tension-sensitive kinetochore protein phosphorylation is the most likely possibility. We combined micromanipulation with immunostaining for phosphoproteins in order to study the effect of tension on kinetochore phosphorylation in mantid spermatocytes. We confirm earlier observations on mammalian cells and grasshopper spermatocytes that misattached chromosomes have phosphorylated kinetochore proteins. We also confirm experiments in grasshopper spermatocytes showing that tension alters kinetochore chemistry: tension from a micromanipulation needle causes kinetochore protein dephosphorylation, and relaxation of tension causes kinetochore protein rephosphorylation. Beyond confirmation, our results provide fresh evidence for phosphorylation as the signal to the checkpoint. First, mantid cells are the only ones in which an effect of tension on the checkpoint has been directly demonstrated; by equally direct experiments, we now show that tension affects kinetochore phosphorylation in these same cells. Second, sex chromosome behavior in mantids provides a natural experiment to test the relationship between phosphorylation and the checkpoint. In grasshoppers, an unpaired sex chromosome is normal, its kinetochore is under-phosphorylated, and the checkpoint is not activated. In mantids, exactly the opposite is true: an unpaired sex chromosome is abnormal, its kinetochore is phosphorylated and, as predicted, the checkpoint is activated. We conclude that tension-sensitive kinetochore protein phosphorylation very likely is the essential link between proper chromosome attachment and the check-point, the link that permits potential errors in chromosome distribution to be detected and avoided.