How mitosis keeps itself in order

How mitosis keeps itself in order C ells progress through mitosis by switching protein activities on and off in a clearly defi ned order. The ubiquitin ligase APC/C deactivates mitotic proteins by targeting them for degradation by the proteasome. The APC/C is activated by two different subunits that recognize short sequence motifs, known as D and KEN boxes, in the target proteins. In early mitosis, once the spindle assembly checkpoint (SAC) has been satisfi ed, the APC/C partners with the activating sub-unit Cdc20 to promote the cell's entry into anaphase. The APC/C then pairs up with Cdh1 to degrade a different set of sub-strates and promote the cell's exit from mitosis. But even substrates targeted by the same activating subunit are degraded in a specifi c sequence. Lu et al. dissect the different ways that mitotic budding yeast achieve this orderly progression (1). Protein degradation during yeast mitosis is typically studied using synchronized cell cultures and Western blotting. " The problem is that the cells rapidly lose synchrony, and you're only looking at the average behavior of the population, " explains David Morgan from the University of California, San Francisco. " We wanted to get a more precise view. " Morgan and colleagues, led by graduate student Dan Lu, therefore followed the disappearance of GFP-tagged APC/C Cdc20 substrates from individual yeast cells by fl uorescence mi-croscopy, an approach that allowed them to measure the timing, rate, and inherent variability of mitotic protein degradation (1). Lu et al.'s analysis revealed that the APC/C Cdc20 initially degrades the S phase cyclin Clb5 and then, six minutes later, targets a protein called securin for destruction , triggering the separation of sister chromatids and the yeast cell's entry into anaphase. " That six-minute difference would be very hard to see by any other method, " Morgan says. Moreover, the precision of the single-cell analysis allowed the researchers to investigate the mechanisms underlying this six-minute gap. Lu and colleagues first examined the role of the SAC, which inhibits the APC/C Cdc20 until the cell's chromosomes are correctly attached to the mitotic spindle. In human cells, the SAC prevents the destruction of securin but has little effect on the degradation of the mammalian S phase regulator , cyclin A. Lu et al. found that Clb5 was degraded a few minutes earlier in yeast lacking a functional SAC, indicating that the checkpoint usually inhibits Clb5 turnover. In …