The metaphase-anaphase transition depends on the robust timing and coordination of multiple processes. By enabling the precise measurement of the dynamics and timing of cohesin cleavage in single cells, our separase biosensor has allowed us to understand this transition in more detail. Our measurements of the timing of separase activity relative to other anaphase events are consistent with a model in which anaphase begins with the abrupt activation of separase, which targets all chromatin-bound Scc1 simultaneously 8 while at the same time inhibiting PP2ACdc55 activity toward Net1 and Scc1. Separase activation, together with increased Scc1 phosphorylation, leads to the rapid cleavage of all cohesin over a period of about one minute. The completion of Scc1 cleavage triggers the initiation of spindle elongation. Because the forces of the spindle are focused at the centromeres, the separated sister chromatids are unzipped from the centromere outwards by the mitotic spindle, whose robust elongation and stability depend on separasedependent Cdc14 activation.
We identified PP2ACdc55
as an inhibitor of mitotic cohesin cleavage in yeast, and our results demonstrate that PP2ACdc55
can counteract the stimulatory phosphorylation of Scc1 by the polo kinase Cdc5. It will be interesting to see whether this regulation is conserved in centromeric cohesin of higher eukaryotes. Vertebrate PP2ARts1
is known to protect centromeric cohesin from the prophase pathway by dephosphorylating the SA/Scc3 subunit (Hauf et al., 2005
; Waizenegger et al., 2000
). However, the phosphatase controlling Scc1 phosphorylation in vertebrates is not known. Given that separase-mediated inhibition of PP2ACdc55
has been demonstrated only in yeast, it is possible that Scc1 dephosphorylation is governed by distinct mechanisms in other species.
We found that dephosphorylation of Scc1 by PP2ACdc55 leads to slower cleavage of both the biosensor and endogenous cohesin by separase. The lack of a change in reporter cleavage rates in cdc55Δ cells is consistent with this hypothesis, as Scc1 should still be phosphorylated during anaphase in these cells. Scc1 phosphorylation might be expected to increase earlier in the cell cycle of cdc55Δ cells, but we detected no gross difference in the timing of Scc1 phosphorylation in a population of cdc55 Δcells released from alpha factor (data not shown). Such experiments are difficult to interpret, however, 9 because of the poor synchrony of the cell population and because cdc55Δ cells display defects in the timing of mitotic entry that could obscure small changes in the timing of Scc1 phosphorylation. Single cell methods with high temporal resolution will be required to allow precise measurements of Scc1 phosphorylation relative to other mitotic events.
Importantly, when PP2ACdc55
activity is rendered insensitive to separase inhibition by deletion of the ZDS
genes, the rate of cohesin cleavage is reduced by half. Thus, we believe that separase activation promotes the phosphorylation of its own substrate by inhibiting the phosphatase that opposes it (). This regulatory relationship could result in a variation of a coherent feedforward circuit that has the potential to filter out background noise in separase activity as well as help govern the timing of cohesin cleavage (Alon, 2007
; Mangan et al., 2003
). Such a filter could reduce the deleterious effects of low levels of spurious separase activity – which would be especially important if this system contains positive feedback loops that amplify small signals into full separase activation.
Consistent with this possibility, we observed an increase in the variability of the rate of cohesin cleavage in cdc55
Δcells (Table S1
). In these cells, the rate and timing of cohesin cleavage depends primarily on the timing of APC/C activation and securin degradation, a relatively slow process lasting about 20 minutes in human cells (Hagting et al., 2002
) and about 10 minutes in yeast (D. Lu and D.O.M., unpublished data). Conversion of this gradual securin destruction process into abrupt cohesin cleavage is likely to depend on noise filters that delay cleavage until some sustained threshold level of separase activity is reached. The control of Cdc55 by separase could provide such a filter. We propose that this circuit enables the timely phosphorylation of Scc1 at anaphase onset and contributes to the remarkable coordination of anaphase events. This regulation is particularly critical in cells lacking securin, where APC/C activation does not contribute to the regulation of separase activation.
It has recently been proposed that Zds1 and Zds2 are stoichiometric subunits of the PP2ACdc55
complex that help regulate its localization and substrate specificity (Rossio and Yoshida, 2011
; Wicky et al., 2010
). Nuclear levels of PP2ACdc55
decline slightly in late mitosis, and deletion of both ZDS
genes results in the accumulation of Cdc55 in the nucleus (Rossio and Yoshida, 2011
). Thus, the Zds proteins appear to promote exclusion of PP2ACdc55
from the nucleus in late mitosis. Forcing Cdc55 into the nucleus inhibits Cdc14 release (Rossio and Yoshida, 2011
), just as we found that forcing Cdc55 localization on cohesin slows cohesin cleavage. These results are consistent with the idea that separase normally inhibits the function of PP2ACdc55
by promoting its nuclear exclusion in a ZDS
-dependent manner. An alternative possibility is that separase and the Zds proteins are not linked in the same pathway, but rather that an increased nuclear concentration of PP2ACdc55
in the zds1/2
mutant simply promotes Scc1 (and Net1) dephosphorylation, leading to the slower dot disappearance we observed and enhanced Cdc14 release (Queralt and Uhlmann, 2008
). We believe the direct linkage model is more likely, based on the previously observed physical interaction between Zds1 and separase (Queralt and Uhlmann, 2008
). Also, separase overexpression cannot trigger the release of Cdc14 in the absence of the Zds proteins, suggesting that they are necessary for separase induced Cdc14 release (Queralt and Uhlmann, 2008
). Further biochemical studies will be required to clarify the regulatory connections between separase and the Zds proteins. In 1 any case, the inhibitory effects of ZDS
gene deletion on cohesin cleavage clearly support our model that PP2ACdc55
counteracts polo phosphorylation of Scc1.
The specificity of the regulatory PP2A subunits towards the Scc1 and Rec8 cohesin subunits is striking. These two regulatory subunits have very distinct folds; while Rts1/B’ has a HEAT-repeat elongated superhelical structure (Cho and Xu, 2007
; Xu et al., 2006
), Cdc55/B is a 7-bladed WD40 propeller (Xu et al., 2008
). Interestingly, substrate specificity appears to be conferred, at least in part, after the regulatory subunit is recruited to the phosphatase, as demonstrated by our fusion proteins. This suggests a role for the regulatory subunit at the level of substrate orientation or catalysis. Indeed, the Rts1/B’ regulatory subunit makes direct interactions with three surface features of the catalytic subunit in close proximity to the active site (Cho and Xu, 2007
; Xu et al., 2006
), while Cdc55/B makes a single interaction (Xu et al., 2008
Our study provides a detailed description of the process of cohesin cleavage. We observed an abrupt onset of separase activation, followed by a constant rate of cohesin cleavage that was completed in approximately one minute. Separase biosensors near the centromere and the telomere were cleaved with similar timing and rates. Interestingly, the cell appears to contain small amounts of separase (under 50 molecules per cell (Ghaemmaghami et al., 2003
), and it is remarkable that this small enzyme population can process all of the established cohesin along the sister chromatids (likely several hundred complexes per cell), as well as cleave at least one additional substrate (Slk19) and control Cdc14 release, in such a synchronous and abrupt manner. It is also intriguing that a prolonged securin degradation process culminates in a relatively sharp activation of separase and cleavage of cohesin. We propose that separase-mediated regulation of 2 PP2ACdc55
activity towards Scc1 contributes to the observed abruptness of cohesin cleavage. We also suspect that future work will lead to the discovery of additional regulatory interactions that enhance the robust and switch-like properties of this fundamental process.