The morphogenesis checkpoint prevents sister chromatid separation independently of Pds1
High levels of a truncated version of the budding yeast p21-activated kinase Cla4 (Cla4t) activate the morphogenesis checkpoint by inhibiting endogenous Cla4 and its paralogue Ste20 (Chiroli et al., 2003
), which share essential functions in bud neck formation, septin ring assembly, and cytokinesis (Johnson, 1999
). Upon CLA4t
overexpression from the GAL1
promoter, haploid yeast cells arrest with wide bud necks, replicated chromosomes, undivided nuclei, short metaphase spindles, and high levels of the securin Pds1 (Chiroli et al., 2003
). In addition, they markedly delay activation of the Polo kinase Cdc5 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200609088/DC1
), suggesting that they arrest in G2.
deletion is sufficient to allow anaphase in most mutants arresting in mitosis, we asked whether it could bypass the G2 arrest caused by high Cla4t levels. Elutriated G1 cells of a pds1Δ
strain with four copies of the GAL1-CLA4t
construct integrated in the genome (4X GAL1-CLA4t pds1Δ
) were released into the cell cycle in the presence of galactose. As expected, DNA replication () and bipolar spindle formation () took place normally in these conditions, whereas bud neck formation was abnormal because of CLA4t
overexpression (not depicted). Surprisingly, pericentromeric chromosomal sequences marked by a tet operator array that binds TetR-GFP (Michaelis et al., 1997
) could not separate in these cells (), indicating that sister chromatid separation did not occur. Nuclear division and spindle elongation did not take place throughout the course of the experiment (), similar to 4X GAL1-CLA4t
cells under the same conditions (). Thus, deletion of PDS1
is not sufficient to bypass the G2 arrest caused by high levels of Cla4t.
Figure 1. G2 arrest by the morphogenesis checkpoint does not depend on securin. (A–C) Strains with the indicated genotypes (ySP3575, ySP3435, and ySP3436) were grown at 25°C in YEPR. Elutriated G1 unbudded cells were released at 25°C in (more ...)
As shown in (D–F), latrunculin-A (Lat-A), which activates the morphogenesis checkpoint by depolymerizing the actin cytoskeleton, induced, like Cla4t, a securin-independent G2 arrest. In fact, cells released from a G1 arrest in the presence of Lat-A did not bud () but replicated DNA () and formed bipolar spindles (). However, neither wild-type nor pds1Δ cells underwent sister chromatid separation, nuclear division, or spindle elongation (). In contrast, the same events took place promptly in the morphogenesis checkpoint–defective swe1Δ cells, which also exited mitosis and entered a new round of DNA replication, as indicated by the appearance of 4C DNA contents (). Altogether, these data indicate that the morphogenesis checkpoint appears to prevent the onset of anaphase independently of securin.
CLA4t overexpression does not impair securin-mediated nuclear import of separase
Besides its inhibitory function, securin also has a positive role in separase activation in several eukaryotic systems, prompting us to test whether Cla4t overproduction might impair Pds1 interaction with the Esp1 separase and/or Esp1 nuclear import. Wild-type, 4X GAL1-CLA4t, and 4X GAL1-CLA4t swe1Δ cells expressing HA-tagged Pds1 (Pds1-HA) and myc-tagged Esp1 (Esp1-myc18) were grown in raffinose, arrested in G1 by α-factor, and released in the presence of galactose, followed by the analysis of Pds1 and Esp1 nuclear localization and physical interaction. As shown in , budding was delayed in 4X GAL1-CLA4t cells compared with wild type, but kinetics of Pds1-HA and Esp1-myc18 nuclear accumulation were similar in the two strains. Thus, Pds1 can still act as an Esp1 molecular chaperone in the presence of high levels of Cla4t. Accordingly, similar levels of Esp1-myc18 were immunoprecipitated with Pds1-HA from both wild-type and 4X GAL1-CLA4t cell extracts ().
Figure 2. Binding to securin and nuclear accumulation of separase are not affected by the morphogenesis checkpoint. Strains with the indicated genotypes (ySP1735, ySP4690, and ySP4692) were grown in YEPR at 25°C, arrested by α-factor, and released (more ...)
Cohesin cleavage is not sufficient for execution of anaphase in the presence of high Cla4t levels
Although lack of securin did not allow chromatid separation upon morphogenesis checkpoint activation, ectopic cohesin cleavage could be expected to trigger nuclear division in the same conditions. We engineered 4X GAL1-CLA4t
cells to express a Mcd1–tobacco etch virus (TEV) variant, where the Esp1 cleavage site at position 268 is replaced by the recognition sequence for the TEV protease (Uhlmann et al., 2000
). We then introduced in the same cells the TEV protease coding sequence under the control of the GAL1
promoter. These cells grow normally under uninduced conditions because the Mcd1-TEV variant can be cleaved by separase at position 180, whereas it is cleaved and fully removed from chromosomes upon TEV induction even if separase is inactive. Small G1 cells of this strain were elutriated and released in the presence of galactose to trigger expression of both Cla4t and TEV. Remarkably, nuclear division did not take place (), suggesting that cohesin cleavage might be insufficient to allow chromosome segregation in 4X GAL1-CLA4t
cells. Conversely, as previously reported (Uhlmann et al., 2000
), cohesin cleavage by the TEV protease was sufficient to trigger anaphase in cells depleted for Cdc20 (), the APC regulatory subunit essential for Pds1 proteolysis and anaphase onset (Peters, 2006
). Thus, cohesin cleavage seems to be sufficient to trigger anaphase in metaphase-arrested cells but not in cells arrested in G2 by the morphogenesis checkpoint.
Figure 3. Mcd1 cleavage is not sufficient for nuclear division upon CLA4t overexpression. (A) GAL1-CLA4t cells expressing Mcd1-TEV and GAL1-TEV (ySP5871) were grown in YEPR at 25°C. Elutriated small G1 cells were released in YEPRG at 25°C (time (more ...)
Because it was formally possible that the lack of nuclear division in 4X GAL1-CLA4t MCD1-TEV
cells was due to inefficient cohesin cleavage, we analyzed the kinetics of cohesin cleavage by the TEV protease in 4X GAL1-CLA4t
versus wild-type cells after release from G1 in the presence of galactose. Full length of Mcd1-TEV tagged with 3 HA epitopes at the C terminus (Mcd1-HA3) and its cleavage product by separase (at position 180) were detectable in both strains in cycling cells and at time 0 (). Upon galactose addition, kinetics of TEV production, as well as appearance of the TEV-induced Mcd1-HA3 cleavage product (at position 268), were similar in the two strains. However, disappearance of full-length Mcd1 and its separase-induced cleavage product, which can both be cleaved by TEV, was slower in 4X GAL1-CLA4t
than wild-type cells (). This might be due to delayed activation of the Polo/Cdc5 kinase, which stimulates Mcd1 cleavage (Alexandru et al., 2001
), in 4X GAL1-CLA4t
versus wild-type cells. In spite of that, most, if not all, Mcd1-HA3 was cleaved by 3 h in 4X GAL1-CLA4t
cells, but nuclear division occurred only in a small fraction of them (). In contrast, >75% of wild-type cells had accomplished nuclear division under the same conditions. Therefore, other mechanisms besides cohesin-mediated sister chromatid cohesion likely contribute to prevent chromosome segregation when the morphogenesis checkpoint is active.
The spindle is functional under morphogenesis checkpoint activation
Because mitotic Cdks regulate spindle assembly and microtubule dynamics, the morphogenesis checkpoint might delay nuclear division through spindle misfunction. Upon bipolar attachment of sister kinetochores to microtubules, spindle forces overwhelm centromeric cohesion, leading to precocious separation of sister centromeres before anaphase (Goshima and Yanagida, 2000
), thus providing a readout for spindle function. We found that sister centromeres of chromosome 15 could separate concomitantly with spindle formation in the presence of Lat-B (), suggesting that spindle forces are normal.
Figure 4. Spindle dynamics is not affected by the morphogenesis checkpoint. (A) cdc24 (ySP305) and cdc24 ndc10-1 (ySP6207) cell cultures were arrested in G1 by α-factor and released at 37°C (time 0). Cells were analyzed at the indicated times for (more ...)
Because kinetochore inactivation by the ndc10-1
mutation prevents kinetochore–microtubule attachment without affecting spindle formation and elongation (Goh and Kilmartin, 1993
), we also asked whether spindle elongation could take place in ndc10-1
cells under morphogenesis checkpoint activation. We induced morphogenetic defects by using a temperature- sensitive cdc24
mutation, which alters a guanine-nucleotide exchange factor for the GTPase Cdc42 that is required for budding (Johnson, 1999
). Upon release of synchronized G1 cells at 37°C, cdc24
cells arrested in G2 as unbudded with undivided nuclei and short metaphase spindles. Lack of kinetochore attachment in cdc24 ndc10-1
cells was sufficient to allow spindle elongation (), suggesting that spindle dynamics is not affected by morphogenetic defects. Therefore, residual sister chromatid cohesion, rather than a misfunctional spindle, is likely responsible for preventing chromosome segregation in the absence of Mcd1 upon morphogenesis checkpoint activation.
The phosphatase PP2ACdc55 prevents sister chromatid separation upon morphogenesis checkpoint activation
Cdc55 is one of the two regulatory subunits of yeast protein phosphatase PP2A and was previously implicated in maintaining sister chromatid cohesion in response to spindle defects (Minshull et al., 1996
). This prompted us to test whether CDC55
deletion could allow sister chromatid separation in Cla4t-overexpressing cells. Elutriated G1 cells of a 4X GAL1-CLA4t cdc55Δ
strain carrying the tetO/tetR-GFP constructs for monitoring sister chromatid separation were released into the cell cycle in the presence of galactose. As shown in , deletion of CDC55
partially rescued the cytokinetic defects caused by high Cla4t levels, indicated by reaccumulation of a small fraction of cells with 1C DNA contents at the end of the first cell cycle. Most cells, however, displayed abnormal bud necks characteristic of 4X GAL1-CLA4t
cells. In spite of that, they underwent efficient sister chromatid separation and nuclear division (), suggesting that Cdc55 prevents anaphase onset when p21-activated kinases are inactive.
Figure 5. PP2ACdc55 prevents sister chromatid separation upon activation of the morphogenesis checkpoint. (A–C) Strains with the indicated genotypes (ySP5115, ySP5112, and ySP5165) were grown at 30°C in YEPR. Elutriated G1 cells were released in (more ...)
Nuclear division could also be induced in 4X GAL1-CLA4t
cells by expressing a mutant form of the Pph21 catalytic subunit (Pph21-L369Δ; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200609088/DC1
) that was shown to preferentially fail to interact with Cdc55 (Jiang, 2006
). Therefore, chromatid cohesion upon morphogenesis checkpoint activation requires the protein phosphatase PP2A bound to Cdc55.
The catalytic and structural PP2A subunits can form mutually exclusive complexes with either one of the regulatory subunits Cdc55 and Rts1 (Evans and Hemmings, 2000
and its human counterpart have recently been shown to prevent precocious dissociation of centromeres both in mitosis and in meiosis I (Kitajima et al., 2006
; Riedel et al., 2006
). In an experiment similar to the one described for cdc55Δ
, we found that pericentromeric sequences could not separate in the majority of 4X GAL1-CLA4t rts1Δ
cells (). When pericentromeric regions did split (~25% of the cells), GFP dots were always found very close to each other () and nuclear division was negligible (), suggesting that PP2ARts1
plays a minor role, compared with PP2ACdc55
, in controlling chromatid cohesion under these circumstances.
Because Cdc55 and Rts1 compete for binding to the other PP2A subunits, sister chromatid separation in the absence of Cdc55 could be ascribed to increased levels of the PP2ARts1 complex. To investigate this possibility, we asked whether 4X GAL1-CLA4t cells lacking both Cdc55 and Rts1 could undergo anaphase. Elutriated G1 cells of the 4X GAL1-CLA4t cdc55Δ rts1Δ strain released in the presence of galactose progressed into the cell cycle very slowly, as a result of budding and replication defects (). In spite of that, those that could finish chromosome replication underwent efficient dissociation of sister chromatids and nuclear division (), suggesting that anaphase onset in 4X GAL1-CLA4t cells lacking Cdc55 is not due to increased levels of PP2ARts1 activity.
We then asked whether PP2ACdc55
also controls sister chromatid cohesion in other conditions that activate the morphogenesis checkpoint. Wild-type and cdc55Δ
cells were arrested in G1 by α-factor and then released in the presence of Lat-A. In these conditions, neither wild-type nor cdc55Δ
cells budded throughout the course of the experiment (). As expected, wild-type cells accumulated with 2C DNA contents, unsevered sister chromatids, undivided nuclei, and short metaphase spindles (). Strikingly, sister chromatids separated efficiently in cdc55Δ
cells under the same conditions, thus allowing spindles to elongate and nuclei to divide (). Finally, because CDC55
deletion causes by itself morphogenetic defects and Swe1 stabilization at low temperatures (Healy et al., 1991
; Yang et al., 2000
), we asked whether the cdc55Δ
mutant could separate sister chromatids at 16°C. At this temperature, cdc55Δ
cells showed prominent morphogenetic defects (not depicted), but nevertheless could split chromatids and divide nuclei, albeit with a delay compared with wild-type cells ().
To directly compare the effects of cohesin inactivation and lack of PP2ACdc55
on sister chromatid separation of cells with morphogenetic defects, we used the temperature-sensitive scc1-73
allele, which inactivates Mcd1 and advances sister chromatid separation relative to wild type at the restrictive temperature (Michaelis et al., 1997
). G1-arrested cdc24
cells either lacking CDC55
or carrying the scc1-73
allele were released at 37°C. cdc24 scc1-73
cells could efficiently separate chromosome V arm sequences, although with a delay compared with scc1-73
cells, but did not elongate spindles or divide nuclei (). In contrast, cdc24 cdc55Δ
cells underwent complete chromosome segregation under the same conditions (). Accordingly, the distance between separating chromatids at 150 min after release was significantly higher in cdc24 cdc55Δ
cells than in cdc24 scc1-73
cells (). Therefore, some residual chromatid cohesion likely persists even when cohesin is inactivated and PP2ACdc55
plays a crucial role in controlling sister chromatid separation when the morphogenesis checkpoint is activated.
Figure 6. Inactivation of PP2ACdc55, but not of cohesin, allows nuclear division in the presence of morphogenetic defects. Strains with the indicated genotypes (ySP601, ySP818, ySP6236, ySP6241, and ySP6214) were arrested in G1 by α-factor at 25°C (more ...)
Mcd1 cleavage does not occur in cdc55Δ cells undergoing anaphase under morphogenesis checkpoint activation
Although ectopic cohesin cleavage did not allow nuclear division during morphogenesis checkpoint activation, CDC55
deletion might still allow anaphase onset in these conditions through cohesin cleavage. To test this possibility, cdc24
, cdc24 swe1Δ
, and cdc24 cdc55Δ
cells were arrested in G1 by α-factor and then released at 37°C, followed by analysis of cell cycle parameters () and Mcd1 cleavage by separase (). As expected, cdc24
cells arrested with 2C DNA contents, unseparated sister chromatids, and metaphase spindles, whereas most cdc24 swe1Δ
cells underwent anaphase and spindle elongation and eventually exited mitosis and rereplicated their chromosomes, accumulating DNA contents higher than 2C (), suggesting that lack of Swe1 overrides cells' ability to sense morphogenetic defects. Interestingly, cdc24 cdc55Δ
cells could also undergo anaphase in the same conditions, albeit with a delay compared with cdc24 swe1Δ
cells, but remained mostly arrested with 2C DNA contents. The Mcd1 cleavage product, which was readily apparent in cdc24 swe1Δ
cells and preceded sister chromatid separation, was mostly negligible in cdc24 cdc55Δ
cells (). Nevertheless, chromatin staining of Mcd1 after chromosome spreading revealed that cohesin remained bound to chromatin in wild-type cells (not depicted) but had dissociated from the chromosomes in nuclei of cdc55Δ
cells that underwent anaphase (). Thus, sister chromatid separation and Mcd1 dissociation from chromosomes in cdc55Δ
cells under morphogenesis checkpoint activation do not seem to correlate with separase-dependent cleavage of cohesin. Accordingly, the Mcd1 cleavage product was not detectable in 4X GAL1-CLA4t cdc55Δ
cells undergoing anaphase in the presence of galactose, similar to 4X GAL1-CLA4t
cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200609088/DC1
), and Mcd1 disappeared from the nuclei of 4X GAL1-CLA4t cdc55Δ
cells in anaphase (Fig. S3 E). Mcd1 displacement from chromatin did not correlate with increased Mcd1 phosphorylation, which could instead be detected as electrophoretic mobility shift in nocodazole-arrested cells (Fig. S3 D). It is interesting to note that SWE1
deletion in Cla4t-overexpressing cells caused rapid Pds1 and Clb2 proteolysis, as well as appearance of the Mcd1 cleavage product, whereas Pds1 and Clb2 remained mostly stable upon deletion of CDC55
Figure 7. Lack of Cdc55 upon Cla4t overproduction allows anaphase in the absence of Mcd1 cleavage. Strains with the indicated genotypes (ySP6249, ySP6250, and ySP6463) growing at 25°C were arrested in G1 by α-factor and released at 37°C (more ...)
Unlike in cdc55Δ
cells under morphogenesis checkpoint activation, sister chromatid separation in nocodazole-treated cdc55Δ
cells was accompanied by Pds1 degradation Mcd1 cleavage, although with a delay compared with the spindle checkpoint–defective mad2Δ
cells (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200609088/DC1
). Therefore, PP2ACdc55
contributes to maintaining sister chromatid cohesion in nocodazole by impinging on the same targets of the spindle assembly checkpoint, as recently suggested by others (Yellman and Burke, 2006
). In contrast, PP2ACdc55
likely prevents sister chromatid separation in G2 through a different mechanism.
Sister chromatid dissociation induced by lack of PP2ACdc55 does not require the Cdc14 phosphatase, Polo kinase, and condensin but can be reversed by topoisomerase II inhibition
has recently been shown to prevent Cdc14 early anaphase release from the nucleolus through Net1 dephosphorylation (Queralt et al., 2006
). Cdc14 can in turn trigger Pds1 proteolysis in nocodazole-arrested cells (Visintin et al., 1998
), and this mechanism has been proposed to be responsible for the precocious dissociation of sister chromatids in nocodazole-treated cdc55Δ
cells (Yellman and Burke, 2006
). We therefore asked whether Cdc14 was released from the nucleolus in cdc55Δ
cells with morphogenetic defects and necessary for their onset of anaphase. Wild-type and cdc55Δ
cells were arrested in G1 by α-factor and released in the presence of Lat-B. In situ immunostaining of Cdc14 showed that anaphase took place in cdc55Δ
cells before Cdc14 release from the nucleolus (). In addition, analysis of cdc55Δ
anaphase cells 150 min after release revealed that a high fraction of them (68.3%; n
= 120) had undergone anaphase with Cdc14 in the nucleolus (), suggesting that premature Cdc14 release is not responsible for sister chromatid separation in these cells.
Figure 8. Cdc14 is not required for nuclear division of cdc55Δ cells with morphogenetic defects. (A) Wild-type (wt; ySP3575) and cdc55Δ (ySP5068) cells were arrested in G1 by α-factor at 30°C and released at 25°C in the presence (more ...)
To test whether Cdc14 was required for the onset of anaphase in cdc55Δ
mutants with morphogenetic defects, we inactivated Cdc14 in cdc24 cdc55Δ
cells with the temperature-sensitive cdc14-3
allele. As a control for Cdc14 inactivation, we analyzed the subcellular localization of the Swi5 transcription factor, whose nuclear import in telophase is strictly dependent on its dephosphorylation by Cdc14 (Visintin et al., 1998
). Cell cultures of cdc24 cdc55Δ
and cdc24 cdc55Δ cdc14-3
strains expressing a myc-tagged Swi5 protein were synchronized in G1 by α-factor and released at 37°C to analyze, over time, budding kinetics, Swi5 localization, and nuclear division. Swi5 was cytoplasmic in both strains throughout most of the cell cycle. However, although it was imported into the nucleus of cdc24 cdc55Δ
telophase cells, it always remained in the cytoplasm of cdc24 cdc55Δ cdc14-3
cells, indicating that Cdc14 had been inactivated (). Lack of Cdc55 allowed a fraction of cdc24
cells to divide nuclei irrespective of Cdc14 function (), indicating that Cdc14 is dispensable for the onset of anaphase in these conditions. Accordingly, Cdc14 was also insufficient to promote sister chromatid separation in CLA4t
- overexpressing cells carrying the dominant TAB6-1
allele, which encodes a hyperactive Cdc14 variant with reduced affinity to its inhibitor Net1 (Shou et al., 2001
Although we did not detect any increase in Mcd1 phosphorylation in cdc55Δ versus wild-type cells overproducing Cla4t (Fig. S3), it was still possible that PP2ACdc55 could prevent sister chromatid separation by counteracting the Cdc5-mediated phosphorylation of a small fraction of Mcd1 or other cohesin subunits. However, inactivation of Cdc5 with the cdc5-2 temperature-sensitive allele did not prevent anaphase in cdc24 cdc55Δ cells (), suggesting that Cdc5 is not required for this process.
Figure 9. Effects of Cdc5, condensin, and topoisomerase II inactivation on anaphase onset in the absence of Cdc55. (A) Strains with the indicated genotypes (W303, ySP305, ySP6130, ySP6146, ySP6121, and ySP6105) were grown in YEPD at 26°C, arrested in G1 (more ...)
Timely sister chromatid segregation, especially of ribosomal DNA and chromosome sequences far from centromeres, depends on condensin and DNA topoisomerase II (DiNardo et al., 1984
; Holm et al., 1985
; Bhalla et al., 2002
; D'Amours et al., 2004
; Sullivan et al., 2004
). We therefore tested the effects of the temperature-sensitive ycg1-10
mutations, affecting condensin and DNA topoisomerase II, respectively, on the unscheduled anaphase of cdc24 cdc55Δ
cells. Although inactivation of Ycg1 had no significant effect, inactivation of topoisomerase II in cdc24 cdc55Δ top2-4
cells mostly prevented anaphase (), suggesting that the presence of topological linkages prevents sister chromatid separation under these conditions. Consistently, the presence of the top 2-4
allele could partially rescue the cold sensitivity of cdc55Δ
cells (), which is presumably due to unscheduled sister chromatid separation in the presence of morphogenetic defects.
We then asked whether morphogenetic defects could arrest the cell cycle in a stage where topological linkages are not resolved, using an assay that allows detection of accumulation of catenated forms of a circular minichromosome (Koshland and Hartwell, 1987
). Unlike top2-4
mutants, however, neither cdc24
() nor GAL1-CLA4t
cells (not depicted) accumulated minichromosome topoisomers. Although we cannot exclude the possibility that the behavior of natural chromosomes is different from that of minichromosomes, the delay of nuclear division caused by the morphogenetic checkpoint does not seem to be accompanied by lack of decatenation.
CDC55 overexpression delays sister chromatid separation independently of Pds1
If PP2ACdc55 acts as an inhibitor of sister chromatid separation, increasing its dosage might delay the onset of anaphase. We therefore introduced into the genome of otherwise wild-type cells multiple copies of a galactose-inducible GAL1-CDC55 construct. Parental and transformed strains growing in raffinose were arrested in G1 with α-factor and released in the presence of galactose. We then monitored separation of the tetO array located 13 kb away from CEN5, as well as spindle formation and elongation (). CDC55 overexpression did not affect bipolar spindle formation but delayed sister chromatid separation, nuclear division, and spindle elongation, causing cells to accumulate in G2. This delay did not depend on functional securin, as PDS1 deletion did not accelerate the onset of anaphase in CDC55-overexpressing cells. High levels of Cdc55 delayed sister chromatid separation at both pericentromeric and telomeric regions (unpublished data), suggesting that PP2ACdc55 prevents dissociation of sister chromatids along their length.
Figure 10. CDC55 overexpression delays anaphase independently of securin. Wild-type (wt; ySP3575), GAL1-CDC55 (ySP5690), and GAL1-CDC55 pds1Δ (ySP5752) strains carrying the tetO/tetR-GFP construct to detect pericentromeric sequences at chromosome V were (more ...)
If PP2ACdc55 acted as anaphase inhibitor independently of securin, we could expect that simultaneous loss of Pds1 and Cdc55 might have additive effects, allowing precocious separation of sister chromatids during the unperturbed cell cycle. Indeed, concomitant deletion of CDC55 and PDS1 turned out to be lethal (unpublished data).