In this paper, we have studied the degradation of securin in dividing human cells. We have used securin FPs to follow the localization and proteolysis of securin in real time. A previous study using a securin–GFP fusion protein in living cells found that securin–GFP transfected into endothelial cells, inhibited mitosis, and promoted apoptosis (Yu et al., 2000
). In those few cells that entered mitosis the protein was judged to disappear in anaphase, but this was based on a qualitative assessment of fluorescence. In contrast, we find that securin–GFP neither induces apoptosis nor inhibits entry into mitosis. Furthermore, we have quantified the disappearance of securin–FP and shown that its degradation begins at metaphase. The differences between our results and the previous study may lie in the cell type used or reflect an advantage of microinjection over transfection.
We have shown that the timing of securin destruction is controlled by the spindle checkpoint; destruction only begins at metaphase, and reimposing the checkpoint in metaphase rapidly inactivates securin proteolysis. Furthermore, disrupting the checkpoint machinery with a dominant negative mutant of Bub1 causes securin to be degraded prematurely, just after nuclear envelope breakdown. Thus, the properties of securin destruction resemble those of cyclin B1. However, securin and cyclin B1 do not have identical subcellular localization patterns in mitosis; unlike cyclin B1, securin does not stain the chromosomes in prometaphase. Therefore, if as suggested previously there is some spatial control on ubiquitination in mitosis (Clute and Pines, 1999
; Huang and Raff, 1999
) this might be able to discriminate between cyclin B1 and securin, although we have not yet found conditions that uncouple securin from cyclin B1 destruction. This is in contrast to budding yeast where securin (Pds1p) and a fraction of the major B-type cyclin, Clb2, are degraded before anaphase, but a significant proportion of Clb2 remains to be degraded later in mitosis (Lim et al., 1998
; Baumer et al., 2000
; Yeong et al., 2000
Although securin destruction begins as soon as the spindle checkpoint is inactivated, sister chromatids often do not separate until much later (~23 min later in PtK1 cells [Rieder et al., 1994
]). Moreover, at anaphase all of the sister chromatids separate with a high degree of synchrony. It is difficult to reconcile this observation with a model in which sister chromatid separation is solely controlled by the activation of separase after securin is destroyed. If this is the case, then active separase could accumulate for ~20 min before sister chromatid disjunction, and it is unlikely that all of the sister chromatids would separate at the same time. Furthermore, the majority of securin−/−
mouse cells must also separate their chromosomes correctly as evidenced by their ability to proliferate in culture (Jallepalli et al., 2001
) and to generate apparently normal animals (Mei et al., 2001
). Thus, we favor a model in which there is a second step to sister chromatid separation, perhaps related to the recent demonstration that the budding yeast separase is only able to recognize the phosphorylated form of its cohesin substrate (Alexandru et al., 2001
). In some cells injected with securin–FP we observe a cut phenotype even after the securin–GFP has fallen below detectable levels. This may be because there is still sufficient securin to inactivate the separase. However, the more interesting possibility is that there is insufficient time for the second step between securin destruction and the cleavage of cohesin.
The second step to sister chromatid separation may involve the inactivation of cyclin B1–CDK1. We find that moderate to high amounts of cyclin B1 () will prevent sister chromatid separation in the absence of securin, and we have shown previously that cyclin A2 must also be degraded to allow anaphase (den Elzen and Pines, 2001
). High amounts of cyclin B–CDK1 activity have been shown to prevent sister chromatid separation in Xenopus
extracts because separase remains phosphorylated and its ability to cleave cohesin in vitro is significantly reduced (Stemmann et al. 2001
). Thus, it appears that cyclin B1–CDK1, and possibly cyclin A2–CDKs, may directly or indirectly inactivate separase. This alternative means of regulating sister chromatid separation could explain why securin is not an essential protein in mammalian cells (Jallepalli et al., 2001
; Mei et al., 2001
Securin degradation is a key event in mitosis. We found that all of the securin mutants that could not be degraded in metaphase block sister chromatid separation, and unlike budding yeast human cells do not have a mechanism to prevent cytokinesis in this event. Thus, the daughter cells inherit an incorrect complement of chromosomes, and this may be the mechanism by which overexpressed human securin transforms 3T3 cells (Pei and Melmed, 1997
). This emphasizes the importance of the spindle checkpoint both to normal progression through mammalian mitosis and for genomic stability. This is underscored by the observations that mice lacking spindle checkpoint components such as Mad2 (Dobles et al., 2000
) and Bub3 (Kalitsis et al., 2000
) die early in embryogenesis with gross mitotic abnormalities. Furthermore, a haplo insufficiency of Mad2 gives rise to chromosomal instability and eventually to tumorigenesis (Michel et al., 2001
) so the level of the checkpoint proteins is likely to be crucial to the proper regulation of securin and cyclin B1 destruction.
Because all of the cells expressing securin mutants that could not be degraded in metaphase exhibit a cut
phenotype, we conclude that degrading human securin is an essential prerequisite for sister chromatid separation. However, the phenotype we observed for the D-box mutant of securin differs from the cut
phenotype observed by Zur and Brandeis (2001)
in which only a small minority (5%) of HeLa cells expressing a nondegradable securin failed to complete cytokinesis and remained connected by chromatin threads. The difference may be due to the different experimental approaches; we microinjected cells and followed them by time-lapse microscopy, whereas Zur and Brandeis (2001)
transfected cells and analyzed them after fixation.
The high rates of whole chromosome loss that we observe for cells with nondegradable securin are similar to the effects of the loss of securin in human cells (Jallepalli et al., 2001
). This might appear paradoxical but can be explained by data indicating that securin is required fully to activate separase (Jallepalli et al., 2001
). Thus, a nondegradable separase inhibitor would have the same effect as an inability to activate separase. Cells without securin also exhibit problems with sister chromatid separation, but these problems are only seen in about a third of the population, perhaps because the cells still have a low but detectable level of active separase.
The behavior of the various securin mutants in mitosis may give some clues to the changes in the machinery underlying progression through mitosis in somatic cells. A securin mutant with a defective D-box but an intact KEN box could be ubiquitinated by APC/CCdh1
but not APC/CCdc20
Although this is an artificial substrate (because normally all the securin should be degraded in metaphase by APC/CCdc20
) in vivo this mutant was stable throughout metaphase but became unstable just before the cell began to elongate at anaphase B. Thus, the switch from APC/CCdc20
- to APC/CCdh1
-dependent destruction appears to happen in anaphase. By extrapolation from results obtained with budding yeast, this could be explained by the disappearance of cyclin B1–CDK activity at the end of metaphase leading to the dephosphorylation of Cdh1. Dephosphorylated Cdh1 would then bind the APC (Kramer et al., 2000
) and begin the degradation of Cdc20 and other KEN box substrates. In support of this model, we find that a nondegradable cyclin B1 mutant prevents the destruction of the securin D-box mutant with an intact KEN box. Alternatively, but less likely, APC/CCdc20
might alter its specificity to recognize the KEN box.
The question arises as to why somatic cells should switch from degradation mediated by APC/CCdc20
in anaphase. Clearly this is not required for exit from mitosis itself because degradation in embryonic cell cycles, such as those of Drosophila
, is mediated solely by Cdc20. It could be that APC/CCdh1
is required because there are some late mitotic regulators that are only present in somatic cells that cannot be recognized by APC/CCdc20
. Alternatively, there may be some proteins that must be degraded in somatic cells but not in embryonic cells. Such proteins could include the regulators or components of the prereplication complex because one of the major differences between somatic cell cycles and embryonic cell cycles is that somatic cells have an interval (G1) between mitosis and the next round of DNA replication. During G1 phase, somatic cells integrate intra- and extracellular signals before commitment to another round of DNA replication rather than the alternative fates of differentiation or quiescence. Thus, it may be important for somatic cells to ensure that components of the DNA replication machinery are not present until they commit to another round of proliferation. Equally, it is becoming clear that once somatic cells exit the cell cycle and differentiate the APC/C plays an important part in the physiology of postmitotic cells (Gieffers et al., 1999
) where it must recognize substrates that are very different from those found in proliferating cells. However, this does not provide an answer to why Cdc20 itself should become a target for degradation upon exit from mitosis in somatic cells.
Lastly, our observation that a second cleavage furrow can form between the separating sister chromatids after all the chromosomes have moved to one pole indicates that the cytokinesis furrow can be very rapidly established, apparently by unseparated chromosomes. It will be interesting to determine which of the chromosomal passenger proteins implicated in cytokinesis are carried toward one spindle pole by the unseparated chromosomes and whether any are left behind at the first cleavage furrow.