A Genetic Screen for Suppressors of the Cytokinesis Checkpoint Defect of SIN Mutants
To further understand the mechanisms controlling the SIN and the cytokinesis checkpoint in fission yeast, we carried out a genetic screen for suppressors of the cytokinesis checkpoint defects in SIN mutants. This screen took advantage of the fact that temperature-sensitive SIN mutants are viable at semipermissive temperatures but that they have reduced SIN function and a defective cytokinesis checkpoint (Mishra et al., 2004
). Due to lack of the checkpoint, these mutations become lethal when the actomyosin ring is slightly perturbed. Thus, these cells fail cytokinesis and they die as multinucleate cells. This can be observed when examining the phenotype of single and double mutants between cdc14-118
(note that S. pombe cdc14
encodes a subunit of the Sid1p kinase and is not a homologue of budding yeast Cdc14) and the type II myosin mutant myo2-E1
. The temperature-sensitive SIN mutant cdc14-118
is viable at 30°C, but it is largely defective for the cytokinesis checkpoint (Mishra et al., 2004
). The temperature-sensitive type II myosin mutant myo2-E1
is also viable at 30°C, but it is slow in completing cytokinesis, and its viability at this temperature depends on the cytokinesis checkpoint (Mishra et al., 2004
). However, the cdc14-118 myo2-E1
double mutant strain is dead at 30°C, because of the combined delay in cytokinesis and defective cytokinesis checkpoint. As shown previously, these cells either fail to make septa, or they make incomplete aberrant septa and become multinucleate (Mishra et al., 2004
The cdc14-118 myo2-E1 strain was screened for spontaneous suppressing mutants at 30°C. From this screen, we identified many spontaneous suppressors, and we picked different-sized colonies, which were backcrossed to wild type. The weaker suppressors only poorly suppressed one of the single mutants, and the stronger suppressors suppressed both cdc14-118 and myo2-E1 (A; data not shown). The best suppressors we identified fell into three complementation groups: group I (6 members: 14, 16, 2-12, 2-13, 3-8, and 4-3), group II (2 members: 6 and 3-3), and group III (1 member: 4-12 called sdc4 for suppressor of defective checkpoint). The first two groups are later referred to as dnt1 and rrn5, respectively (for reasons, see Materials and Methods). Outcrossing revealed that although the dnt1 mutant cells showed no obvious growth defect, the two alleles of rrn5 were temperature sensitive on their own, and the sdc4-12 strain grew slowly at all temperatures (A).
Figure 1. Suppressors of cdc14-118 myo2-E1 double mutant. (A) Selected suppressor single mutants and triple mutants with cdc14-118 myo2-E1 were grown in YE at 25°C, and then serial dilutions were spotted on YE plates. Plates were incubated at different (more ...)
Examination of the cdc14-118 myo2-E1 cells carrying the different suppressor mutations showed that they all had similar morphology and that they had recovered the ability to form complete septa at 30°C (; data not shown). To characterize the phenotype more closely, we examined cdc14-118 myo2-E1 and cdc14-118 myo2-E1 dnt1 cells after shift from 25 to 30°C for 4 h. In the cdc14-118 myo2-E1 cells, the number of mononucleate cells decrease and the cells become bi- and tetranucleate presumably due to cytokinesis defects caused by their inability to make proper septa (). In contrast, the cdc14-118 myo2-E1 dnt1 cells seem to have at least partially restored cytokinesis checkpoint function, because these cells show a delay as binucleate cells but eventually can divide, because they maintain a mononucleate population and do not accumulate tetranucleate cells (A). These cells also recover the ability to make proper septa (B).
Figure 2. dnt1Δ promotes proper completion of cytokinesis in the cdc14-118 myo2-E1 cells. Asynchronous cells of the indicated genotypes were grown at 25°C to log phase cell density, and portions were shifted to 30°C for 4 h. Cells were methanol (more ...)
Suppressors Encode Nucleolar Proteins
The group II suppressor gene was cloned by complementation of its temperature-sensitive phenotype and determined to be the rrn5+
gene (see Materials and Methods
). The rrn5+
gene encodes an upstream activating factor (UAF) for RNA polymerase I involved in transcription of rRNA (Liu et al., 2002
). Examination of the phenotype of the rrn5-S6
mutant at restrictive temperature revealed an unusual nuclear architecture. In wild-type cells, the nuclear DNA forms a crescent shape on one side of the nucleus, with the nucleolus occupying the other portion of the nucleus. However, at its restrictive temperature of 36°C the rrn5-S6
cells often had a ring-shaped nuclear DNA pattern (B). The only other mutants reported to date with this phenotype are the topoisomerase I and II double mutants, and the temperature-sensitive mutant nuc1-632
, which carries mutation in the largest subunit of RNA polymerase I (Hirano et al., 1986
). Interestingly, the sdc4-12
mutant also displays the ring-shaped nuclear DNA phenotype (B), which, together with genetic analysis described below, suggests that it might also affect RNA polymerase I transcription. Both point mutations and null mutations in the dnt1
did not cause the ring-shaped chromatin phenotype. To determine the localization of Rrn5, we expressed plasmid-borne and GFP-tagged Rrn5 in wild-type cells. Not surprisingly, similar to Nuc1, Rrn5 showed nucleolar localization as judged by colocalization with the non-DAPI staining region of the nucleus (A).
Figure 3. Nucleolar localization of suppressor proteins. The indicated strains were grown at 30°C. (A) Cells transformed with pREP42-rrn5+-GFP were first grown in EMM with thiamine (repressed), and then they were induced for 18 h in EMM without thiamine. (more ...)
To investigate whether general perturbation of the RNA polymerase (Pol) I transcription complex can suppress cdc14-118 myo2-E1 cells, we tested whether the nuc1-632 could also rescue this double mutant at 30°C. Although the nuc1-632 cells grew very poorly on their own at 30°C, the nuc1-632 mutation could weakly rescue cdc14-118 myo2-E1 cells at this temperature (A).
The group I suppressors turned out to be in the dnt1 gene that had been identified in our laboratory as part of an unrelated proteomics screen by using mass spectrometry (see Materials and Methods; Jin and McCollum, unpublished observations). dnt1Δ deletion mutants were viable, and they grew at rates similar to wild-type cells. The dnt1Δ mutation also rescued the growth defect of cdc14-118 myo2-E1 cells at 30°C (A). Interestingly, Dnt1 is also a nucleolar protein, because Dnt1-GFP is localized in the nucleolus as two or more punctate dots throughout the cell cycle (, B and D). Dnt1-GFP signals can also be observed faintly in the rest of the nucleoplasm. In late anaphase, Dnt1 localizes to the ends of the mitotic spindle (C).
Functional Interdependence of Dnt1 and Suppressors Involved in rDNA Transcription
Three of our suppressors, rrn5-S6, nuc1-632, and sdc4-12, showed a characteristic ring-shaped DNA phenotype, and they all grew slowly even at permissive temperature, probably due to reduced rDNA transcription. Although dnt1Δ mutants did not have a reduced growth rate or defects in nucleolar positioning, genetic analysis suggested that they may also have a role in RNA Pol I transcription. Double mutant analysis revealed that all combinations of double mutants between nuc1-632, rrn5-S6, sdc4-12, and dnt1Δ resulted in either synthetic lethality or very sick and slow-growing cells (). These data suggest that Dnt1 and Sdc4 might function in rDNA transcription like Rrn5 and Nuc1.
Negative genetic interactions between suppressorsa
Further evidence also suggested an interaction between RNA Pol I and Dnt1. First, localization of Dnt1 to the nucleolus was disrupted in rrn5-S6, sdc4-12, and nuc1-632 cells (A). At 30°C, the temperature where all these mutations can suppress the growth defects of cdc14-118 myo2-E1, Dnt1 is not enriched in the nucleolus in these mutants; instead, it was found in the nucleoplasm surrounding the nucleolus. This effect was not due to a global disruption of the nucleolus, because the nucleolar protein Gar2 still localized normally to the displaced nucleolus in these mutants at 30°C (Supplemental Figure S1). This disrupted localization of Dnt1 was observed in rrn5-S6 and nuc1-632 mutants even at 25°C, the permissive temperature for these mutants (data not shown). We also found that Dnt1 is required for maintaining the exclusive nucleolar localization of Nuc1, because examination of Nuc1-GFP localization in dnt1Δ cells showed signals not only in nucleolus but also in nucleoplasm (B). This localization pattern is distinct from wild-type cells, in which Nuc1-GFP is almost exclusively found in nucleolus (B). We noticed that Nuc1-GFP dnt1Δ cells grew very poorly, although strains carrying either individual allele grew well. This might be because GFP-tagged Nuc1 is not completely functional; thus, nuc1-GFP dnt1Δ cells demonstrate a negative genetic interaction (data not shown). Consistent with the genetic interactions we observed between all suppressors, we also found that nucleolar Nuc1-GFP localization is slightly disrupted in rrn5-S6 and sdc4-12 mutants. Like in dnt1Δ cells, strong Nuc1-GFP signals were found at the periphery of the nucleolus and weaker signals in the nucleoplasm (B).
Figure 4. Nucleolar localization of Dnt1 and Nuc1 is disrupted in suppressor mutants. Wild-type and mutant cells with the indicated genotypes were first grown at 25°C, and then they were shifted to 30°C for 4 h before being visualized by fluorescence (more ...)
S. pombe Dnt1: A Homologue of Budding Yeast Net1/Cfi1?
So why might these mutations in nucleolar proteins suppress cdc14-118 myo2-E1
? A similar screen in S. cerevisiae
identified Net1/Cfi1, the nucleolar inhibitor of the Cdc14 phosphatase (Shou et al., 1999
). Our basic database searches had not revealed any obvious homologues of Net1/Cfi1 in S. pombe
. However, in the course of this study, more sophisticated database searching (with advice from Dr. Aaron Neiman, SUNY, Stony Brook, NY) identified a candidate S. pombe
protein called SPBC25D12.02c, which corresponds to Dnt1. Among Net1/Cfi1 homologues in related budding yeast, only the N-terminal 180 amino acids are conserved. This region is also conserved in a second nucleolar protein in S. cerevisiae
called Tof2 (Huang et al., 2006
) (A). Psi-Blast searches with this region identified SPBC25D12.02c as the best homologue in S. pombe
of the S. cerevisiae
NET1/CFI1 and TOF2 genes (A).
Figure 5. Relationship between S. pombe Dnt1 and S. cerevisiae Net1/Cfi1. (A) Sequence alignment between Dnt1, Net1, and Tof2 at the N termini of the three proteins. (B) Rescue of cdc14-118 by dnt1Δ can be reversed by expression of budding yeast NET1. dnt1 (more ...)
The similarity of Dnt1 to Net1/Cfi1 is intriguing, because both Dnt1 and Net1/Cfi1 were identified in similar screens for suppressors of the SIN and MEN pathways, respectively. As described above, we found that dnt1Δ rescues not only the cdc14-118 myo2-E1 double mutant but also either single mutant (). Further study showed that dnt1Δ can weakly suppress other SIN mutants (), consistent with Dnt1 being an inhibitor of the SIN. To examine functional conservation between Net1/Cfi1 and Dnt1, we tested whether Net1/Cfi1 could reverse the effects of dnt1Δ on the SIN. We had found that dnt1Δ partially rescues the growth defect of the cdc14-118 mutant, allowing it to grow at 33°C (). We expressed Net1/Cfi1 from plasmids (pREP41X-NET1-GFP, pREP3X-NET1-GFP) under the control of inducible nmt1 promoter in dnt1Δ cdc14-118 cells, and we observed that these cells are dead at 33°C, whereas cells with control plasmid grow well, showing that Net1/Cfi1 expression reversed the rescue phenotype of dnt1Δ at this temperature (B). It is possible that this simply reflects toxicity associated with expression of NET1/CFI1 in S. pombe. However, Net1/Cfi1 expressed from either strong (pREP3X) or intermediate (pREP41X) nmt1 promoters gives a similar reversion phenotype; also, the reversion of suppression occurred even in the presence of thiamine when only low levels of Net1/Cfi1 are expressed. Furthermore, Net1/Cfi1 expression did not inhibit growth at lower temperatures, showing that at these expression levels it was not acting as a general growth inhibitor.
Summary of rescue of SIN mutants by dnt1 Δ
Other similarities between Dnt1 and Net1/Cfi1 include the fact that both proteins localize to the nucleolus (), and like Net1/Cfi1 (Straight et al., 1999
; Shou et al., 2001
), we found that Dnt1 is required for rDNA silencing as judged by increased expression (i.e., derepression) of a reporter gene (ura4+
) integrated into the rDNA repeats (Thon and Verhein-Hansen, 2000
) (C). Although we have not tested directly whether other suppressors have a similar effect on rDNA silencing, it would not be surprising if they did because RNA Pol I transcription activity is required for rDNA silencing in S. cerevisiae
(Shou et al., 2001
). Additionally, as with Net1/Cfi1 (Shou and Deshaies, 2002
), Dnt1 is also involved in maintenance of minichromosomes. dnt1
Δ cells showed an almost 100-fold increase in minichromosome loss rate compared with wild-type cells: with loss rate of 1.78 × 10−2
Δ cells versus 2 × 10−4
in wild-type cells.
Although the genetic interactions we observe between dnt1
Δ and mutants in the RNA polymerase I machinery suggest that Dnt1 may participate in Pol I transcription like Net1/Cfi1, we do not think that it plays a direct role, because unlike the net1
Δ mutant, dnt1
Δ cells do not have reduced growth rates compared with wild-type cells. Additionally, we did not observe cross-complementation between Dnt1 and Net1/Cfi1 for their putative roles in Pol I transcription. Specifically, we found that Net1/Cfi1 could not rescue the synthetic growth defects we observed in dnt1
strains (data not shown). We also tested whether Dnt1 could rescue the growth defects of net1
Δ cells at high temperatures, which are thought to be due to defects in Pol I activity, because they can be rescued by overexpression by Pol I transcription factors (Shou et al., 2001
). However, Dnt1 was not able to rescue the growth defects of net1
Δ cells at high temperatures (data not shown). In summary, although we found some interesting similarities between Dnt1 and Net1/Cfi1, the proteins do not seem to be functionally interchangeable.
Does Dnt1 Act by Antagonizing Clp1?
We next tested whether Dnt1 and Net1/Cfi1 inhibit the SIN and MEN signaling pathways, respectively, through a common mechanism. It is known that Net1/Cfi1 inhibits MEN signaling by binding to the Cdc14 phosphatase, and both sequestering it in the nucleolus and inhibiting its phosphatase activity (Traverso et al., 2001
). Although we were able to detect a modest interaction between Dnt1 and Clp1 in the yeast two-hybrid assay (D), we have been unable to detect an interaction between endogenous or bacterially expressed Dnt1 and Clp1 by coimmunoprecipitation or in vitro binding assays (data not shown). In addition, bacterially expressed Dnt1 does not seem to inhibit Clp1 phosphatase activity in vitro (Ray and McCollum, unpublished data).
dnt1Δ and the Other Suppressors Do Not Cause Premature Release of Clp1 from Nucleolus
Because dnt1+ and the other suppressors we identified encode nucleolar proteins like Clp1, we thought that the suppressors might act by causing release of Clp1 from the nucleolus and allowing it to remain active and promote cytokinesis checkpoint signaling. To address whether the suppressors we isolated have effects on nucleolar localization of Clp1, we examined the localization of Clp1-GFP in dnt1Δ, rrn5-S6, sdc4-12, and nuc1-632 mutant strains (A). All cells were first grown at 25°C, and then they were shifted to 30°C for 4 h, the temperature at which they showed suppression of cdc14-118 myo2-E1. Except for dnt1Δ mutant, all the other mutants show a characteristic ring-shaped DNA phenotype at permissive or restrictive temperature, with the nucleolus in the center of the nucleus. However, we did not find Clp1 to be released prematurely in interphase cells in any of the suppressor mutants, even at fully restrictive temperature (A; data not shown). We also compared Clp1-GFP localization in cdc14-118 myo2-E1 and cdc14-118 myo2-E1 dnt1Δ cells to myo2-E1 cells after shift to 30°C. By comparing the ratio of the mean intensity of nucleolar verses cytoplasmic fluorescence in telophase cells we found, as expected, that Clp1 remains out of the nucleolus in myo2-E1 cells (B) as normally occurs when cytokinesis is perturbed and the cytokinesis checkpoint is activated. However, Clp1-GFP returned to the nucleolus prematurely in both cdc14-118 myo2-E1 and cdc14-118 myo2-E1 dnt1Δ cells (B). Together, these data indicate that the suppressors of the cdc14-118 myo2-E1 double mutant do not rescue by simply keeping Clp1 out of nucleolus.
Figure 6. (A) Suppressors do not cause premature release of Clp1 from nucleolus. Wild-type and mutant cells with indicated genotypes were first grown at 25°C, and then they were shifted to 30 or 36°C for 4 h before being fixed and stained with DAPI. (more ...)
Dnt1 Can Regulate the SIN Independently of Clp1
As described above, we found that dnt1Δ rescues not only the cdc14-118 myo2-E1 double mutant but also either single mutant as well as other SIN mutants. If the rescue of SIN mutants by dnt1Δ was through Clp1, then it should depend on clp1+. To test this, we compared the phenotypes of different combinations of single, double, and triple mutants between cdc14-118, clp1Δ, and dnt1Δ (). We found that cdc14-118 cells can grow up to 30°C but that they die at the restrictive temperature of 33°C. As expected, deletion of Clp1 and Dnt1 have opposite effects on the cdc14-118 mutant, with clp1Δ reducing the restrictive temperature of cdc14-118 cells to 30°C and dnt1Δ raising the restrictive temperature of cdc14-118 cells to 36°C. Interestingly, deletion of dnt1+ raises the restrictive temperature of the cdc14-118 clp1Δ double mutant from 30 to 33°C, showing that Dnt1 can affect the SIN even in the absence of clp1+. We also found that dnt1Δ could promote growth of the myo2-E1 mutant in the absence of Clp1 (data not shown). Together, these results clearly show that dnt1Δ can promote SIN function independent of Clp1, and the simplest interpretation of our results is that Clp1 and Dnt1 act on the SIN independently.
Figure 7. Dnt1 can regulate the SIN independently of Clp1. Wild-type and mutant cells with indicated genotypes were first grown in YE at 25°C, and then they were serially diluted and spotted on plates of YE. Plates were incubated at different temperatures (more ...)
Another piece of evidence supporting the idea that Dnt1 can regulate SIN signaling and cytokinesis checkpoint in the absence of Clp1 came from our analysis of checkpoint activation and SIN signaling in dnt1
Δ and clp1
Δ single and double mutant cells. These cells also expressed Cdc7-GFP whose localization to the SPB can serve as a marker for SIN activation (for review, see McCollum and Gould, 2001
). A low dosage of latrunculin, a drug that prevents actin polymerization (Ayscough et al., 1997
), has been shown to be able to activate the cytokinesis checkpoint in S. pombe
by slowing down actomyosin ring contraction (Mishra et al., 2004
). We compared the kinetics of nuclear cycle progression in synchronized wild-type, dnt1
Δ, and clp1
Δ cells upon treatment with 4 μM latrunculin B in liquid cultures (A). Wild-type and dnt
Δ cells, which have an intact checkpoint, remained binucleate with the SIN activated, and they slowly formed a septum (, A and B). In contrast, clp1
Δ cells, which lack the checkpoint, are unable to maintain SIN signaling, and they become multinucleate and are unable to form complete septa (, A and B) (Mishra et al., 2004
). Unlike clp1
Δ single mutants, clp1
Δ cells maintain SIN signaling and remain blocked as binucleate cells for an extended period, although not as long as wild-type or dnt1
Δ cells (A). These data suggest that dnt1
Δ allows cells to maintain SIN signaling and keep the cytokinesis checkpoint active even in the total absence of Clp1. Therefore, Dnt1 must be able to affect SIN signaling through an alternative pathway.
Figure 8. dnt1Δ has a positive effect on SIN signaling even in the absence of Clp1. Wild-type and mutant cells with the indicated genotypes (all carrying Cdc7-GFP) were first grown in YE at 30°C, and then they were synchronized in early G2 by centrifugal (more ...)