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Vol. 20, Issue 16, 3646-3659, August 15, 2009
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*Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; and Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, KY 40536
Submitted April 9, 2009;
Revised June 18, 2009;
Accepted June 19, 2009
Monitoring Editor: Daniel J. Lew
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Highly intricate mechanisms control these processes, with various macromolecular complexes coordinating their activities such that the integrity of cell division is maintained. The chromosomal passenger complex (CPC), which is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin, functions as one of these critical regulatory complexes. As its name implies, this complex travels on chromosomes to various sites during cell division such that it can execute specific tasks at distinct locations and times (Earnshaw and Bernat, 1991). These functions include, but are not limited to, chromosome condensation, stabilization of the mitotic spindle, correction of improper kinetochore-microtubule attachments, and regulation of cytokinesis (for reviews, see Vagnarelli and Earnshaw, 2004; Vader et al., 2006; Ruchaud et al., 2007).
The four CPC subunits are highly interdependent for proper localization, activity, and stability (Ruchaud et al., 2007). Consistent with such interdependency, these subunits are widely conserved among eukaryotes (Ruchaud et al., 2007). However, it has proven difficult to identify Borealin homologues in yeasts. Though some have speculated that yeast Borealin homologues might not exist because of fusion of the Borealin and Survivin subunits into a single yeast Survivin homologue (Vader et al., 2006), recent evidence suggests that there are in fact yeast Borealin homologues (Nakajima et al., 2009). Nonetheless, a Borealin homologue in the fission yeast, Schizosaccharomyces pombe, has not been characterized.
Similar to the CPC, Cdc14-family phosphatases are also significant regulators of cell division. Named for Cdc14, the founding member identified in the budding yeast, Saccharomyces cerevisiae, these phosphatases reverse Cdk-mediated phosphorylation events to promote mitotic exit and cytokinesis (Queralt and Uhlmann, 2008). Like other Cdc14 family members, the S. pombe Cdc14 homologue, Clp1/Flp1, also functions in these processes. Specifically, Clp1 reverses Cdk1-mediated phosphorylation of the mitotic inducer Cdc25, allowing degradation of Cdc25 by the anaphase-promoting complex/cyclosome at the end of mitosis and ending the Cdk1 auto-amplification loop (Esteban et al., 2004; Wolfe and Gould, 2004). Furthermore, Clp1 associates with the contractile ring (CR) scaffold protein Mid1, and, through this interaction, Clp1 enhances CR stability and the precision of cytokinesis (Clifford et al., 2008).
In addition to these crucial functions during the concluding stages of the cell cycle, Cdc14-family phosphatases regulate chromosome segregation and CPC function during mitosis. In S. pombe, Clp1 associates with the S. pombe Aurora B kinase homologue, Ark1, and deletion of clp1 results in increased cosegregation of sister chromatids (Trautmann et al., 2004). In S. cerevisiae, Cdc14 regulates localization of the CPC to the spindle during anaphase (Pereira and Schiebel, 2003; Stoepel et al., 2005). This Cdc14-mediated targeting of the CPC is crucial to its subsequent recruitment of the separase Esp1 and the chromosomal passenger Slk19 (Pereira and Schiebel, 2003; Khmelinskii et al., 2007) that influence the formation and stabilization of the mitotic spindle and the spindle midzone (Zeng et al., 1999; Jensen et al., 2001). Yet, although it is clear that Cdc14-family phosphatases regulate CPC localization and function, a reciprocal relationship has not been described.
Though effects of the CPC on Cdc14-family phosphatases are unclear, the CPC is known to play a critical role in cytokinesis in many organisms (Carmena, 2008). For example, in S. cerevisiae, CPC subunits regulate septin dynamics (Gillis et al., 2005; Thomas and Kaplan, 2007), and the Aurora B homologue, Ipl1, mediates a NoCut checkpoint that delays cytokinesis when chromatin is stalled in the cleavage plane (Norden et al., 2006; Mendoza et al., 2009). Interestingly, Aurora B functions in a similar checkpoint in human cells, inhibiting abscission in the presence of unsegregated DNA (Steigemann et al., 2009). However, in S. pombe, the contribution of the CPC to cytokinesis is thought to be negligible. Overexpression of kinase-dead Ark1 does not affect cytokinesis but instead results in cut phenotypes in which septa form through unsegregated DNA (Petersen and Hagan, 2003), and ark1 temperature-sensitive mutants similarly exhibit cut phenotypes. Because the division machinery appears intact in cut cells, these data suggest that Ark1 does not play a critical role in S. pombe cytokinesis (Petersen and Hagan, 2003). As a result, connections between the CPC and the S. pombe cytokinetic apparatus have not been further explored.
Through our studies of Clp1-associated proteins in S. pombe that will be discussed in detail elsewhere, we identified a Borealin-like protein, Nbl1. Here, we characterize Nbl1 as an S. pombe Borealin homologue by describing its CPC associations, its relevance to Ark1 activity and stability, its localization during the cell cycle, and its sequence and proposed structural similarity to human Borealin. Our analysis of the Nbl1-Clp1 association reveals that CPC activity is required for proper accumulation of Clp1 at the CR. We also present additional genetic evidence that the CPC influences cytokinesis. This study confirms the presence of a Borealin homologue in S. pombe and provides data linking the CPC and the process of cytokinesis in S. pombe.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Standard cloning methods were used to generate the mApple:natR cassette from a plasmid generously provided by Dr. Ryoma Ohi (Vanderbilt University, Nashville, TN). A lithium acetate method was used in S. pombe tagging transformations (Keeney and Boeke, 1994), and integration of tags was verified using whole-cell PCR or fluorescence microscopy. Introduction of tagged loci into other strains was accomplished using standard S. pombe mating, sporulation, and tetrad dissection techniques. The nbl1(1-69) truncation was established in a wild-type diploid strain through the introduction of a stop codon and kanR cassette after proline 69 via tagging. Expression of nmt41 nbl1(1-95) was controlled in nbl1-shutoff strains by the addition or removal of 5 µg/ml thiamine to the medium. For block of nda3-KM311 strains in prometaphase, cells were grown at 32°C and then shifted to 17°C for 7 h. Subsequent release of these cells to the permissive temperature at 32°C allowed for synchronous progression into anaphase.
For spot assays, cells were grown to midlog phase at 25°C, 10 million cells were resuspended in 1 ml water, and 1 ml serial dilutions were made. 2.5 µL of each dilution were plated on YE, YE plus DMSO alone, or YE plus low dose (0.2 µM) latrunculin A in DMSO. Plates were incubated at 25°C, 27°C, 29°C, or 32°C for 4 to 5 d.
nbl1 Disruption, Shutoff, and Overexpression
Disruption of nbl1+ was achieved by PCR-based one-step homologous recombination (Bahler et al., 1998). Specifically, the nbl1(1-95) fragment was targeted for deletion using ura4+ as the selectable marker. After transformation of an ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32 h-/h+ diploid with the relevant amplified fragment, stable integrants were selected and the nbl1(1-95) deletion was confirmed by PCR. Tetrad dissection of this diploid strain (KGY6731) was performed using standard techniques. For spore germination experiments using KGY6731, sporulating diploids were digested with glusalase at 32°C, allowed to recover in YE at 32°C for 1 h, and then grown in EMM plus adenine and leucine overnight at 25°C.
A pZERO-2 vector with nbl1(1-95) cDNA was generated by Integrated DNA Technologies. nbl1(1-95) cDNA was excised from this vector and cloned into pREP41, placing nbl1(1-95) expression under control of pREP41's thiamine-repressible nmt41 promoter (Basi et al., 1993). The nmt41-nbl1(1-95) fragment was subcloned into a pJK148 vector, linearized within the leu1 gene with NruI, transformed into KGY247, and an integrant at the leu1 locus was identified (KGY7846). In addition, a pSK vector with nbl1(1-95) cDNA and flanking sequences was constructed, and nbl1(1-95) cDNA and flanking sequences were excised from this vector and inserted into a pIRT2 vector. KGY6731 was then transformed with this plasmid, and transformed KGY6731 was sporulated in nitrogen-lacking EMM. Spores were subsequently isolated using glusalase, and nbl1-disrupted cells covered by the plasmid (KGY7841) were selected on EMM plus adenine. KGY7841 was then crossed to KGY7846, and KGY7919, a nbl1-shutoff strain which can grow on EMM plus adenine but not on EMM plus adenine and thiamine, was selected. A pIRTSMART vector with nbl1+ cDNA was also generated by Integrated DNA Technologies. nbl1+ cDNA was excised from this vector and cloned into pREP1, placing nbl1+ expression under control of pREP1's thiamine-repressible promoter (Maundrell, 1990, 1993).
Construction of the nbl1 Phosphosite Mutant
The nbl1-T91A mutation was introduced by site-directed mutagenesis into the pIRT2 vector containing nbl1(1-95) cDNA and flanking sequences. KGY6731 was then transformed with this plasmid, and haploid nbl1-disrupted cells covered by the plasmid were selected. This strain (KGY8048) was then grown to midlog phase in YE, and 10 million cells were plated on YE plus 5-FOA to select for the appropriate replacement strain, which was confirmed by PCR and sequence analysis.
Protein Methods and Mass Spectrometry
Cells were lysed by bead disruption in NP-40 lysis buffer in either native or denaturing conditions as previously described (Gould et al., 1991), except with the addition of 0.1 to 0.5 mM diisopropyl fluorophosphate (Sigma–Aldrich, St. Louis, MO). Proteins were immunoprecipitated by anti-Myc (9E10), anti-GFP (Roche), or anti-FLAG (M2; Sigma–Aldrich) antibodies. For comparison of the Ark1-GFP levels from different genetic backgrounds, an aliquot of cell lysates was taken to detect Cdc2 levels (using anti-PSTAIR) and protein levels were adjusted before performing immunoprecipitations. Immunoblot analysis was performed as previously described (Wolfe et al., 2006) except that secondary antibodies were conjugated to Alexa Fluor 680 (Invitrogen, Carlsbad, CA) or IRDye800 (LI-COR Biosciences) and visualized using an Odyssey scanner (LI-COR Biosciences, Lincoln, NE). Purification of Clp1-C286S-TAP and Nbl1(1-95)-TAP and subsequent identification of Clp1- and Nbl1-interacting proteins by mass spectrometry were performed as previously described (Gould et al., 2004; Roberts-Galbraith et al., 2009).
In Vitro Kinase Assays
The Ark1 kinase assay was performed as previously described (Petersen et al., 2001; Ohi et al., 2004) with minor modifications. Briefly, anti-GFP immunoprecipitates were washed three times in 1 ml NP40 buffer and three times in 1 ml 1x kinase buffer (20 mM K-HEPES, pH7.8, 5 mM MgCl2, 1 mM EGTA, and 1 mM DTT). After washes, liquid was aspirated and 4 µL 5x kinase buffer was added to resuspend the beads. 5 µg purified Histone H3.2 (New England Biolabs), 50 µM cold ATP, and 5 µCi of [
-32P] ATP (GE Healthcare, Piscataway, NJ) were added. After incubation with shaking at 30°C for 30 min, reactions were terminated by addition of 5 µl of 5x SDS-PAGE loading buffer, and proteins were resolved on a 4% to 12% Bis-Tris gel (Invitrogen). The gel was cut in half and the upper portion (30 to 90 kDa) was transferred to a PVDF membrane and blotted with anti-GFP antibodies to detect Ark1-GFP. The lower part (10 to 30 kDa) of the same gel was stained by Coomassie blue dye to visualize Histone H3.2, and, after drying of the gel, autoradiography was used to check incorporation of 32P.
For the Cdk1 kinase assay, 
50 ng of recombinant Cdk1 kinase complex, purified from baculovirus-infected insect cells as previously described (Yoon et al., 2002), was incubated with 200 ng of bacterially produced His6-Nbl1(1-95) in a reaction buffer containing 50 mM Tris pH 7.4, 10 mM MgCl2, 2 µM DTT, 10 µM unlabeled ATP, and 5 µCi of [-32P] ATP. After incubation with shaking at 30°C for 30 min, reactions were terminated with 5x sample buffer, and samples were boiled and separated by SDS-PAGE. Coomassie blue staining and autoradiography were subsequently performed.
Microscopy
For live-cell microscopy, wild-type cells, unless otherwise noted, were grown to midlog phase at 25°C and imaged. Temperature-sensitive strains were first grown to midlog phase at 25°C, synchronized in G2 phase using a 7% to 30% lactose gradient, and shifted to 36°C for 2 to 3 h before live-cell microscopy. All live images were acquired using a spinning disk confocal microscope (Ultraview LCI; PerkinElmer, Foster City, CA) equipped with a 100x NA 1.40 Plan-Apochromat oil immersion objective, a 488-nm argon ion laser (GFP), and a 594-nm helium neon laser (RFP, mCherry, mTomato, mApple). Images were taken via a charge-coupled device camera (Orca-ER; Hamamatsu Photonics) and subsequently processed using Metamorph 7.1 software (MDS Analytical Technologies, Molecular Devices, Sunnyvale, CA). Z-section slices were 0.5 µm. Time-lapse images were taken of cells on YE agar pads sealed with Valap (a Vaseline, lanolin, and paraffin mixture). An objective heater system (Bioptech) was used to maintain the restrictive temperature during imaging of temperature-sensitive strains.
To visualize DNA or cell walls, cells were fixed in 70% ethanol and stained with DAPI or methyl blue. Images were acquired using a microscope (Axioskop II; Carl Zeiss, Thornwood, NY) equipped with a 100x NA 1.40 Plan-Apochromat oil immersion objective and a halogen lamp. OpenLab 4.0.3 software (PerkinElmer) was used to process these images.
Images of yeast colonies were acquired by focusing a camera (PowerShot SD750; Canon, Tokyo, Japan) through a microscope (Universal; Carl Zeiss) equipped with a 20x NA 0.32 objective.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For simplicity, we will subsequently refer to SPBC725.12 as Nbl1 (novel Borealin-like 1) based on the studies described in this article.
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) and were not observed in wild-type cells (Supplementary Figure 1B). Thus, Nbl1 appears to affect chromosome segregation.
To verify that the observed cut phenotypes are in fact due to nbl1 malfunction, we rescued the null mutation with a genomic clone containing nbl1+ cDNA (data not shown) and also developed a nbl1-shutoff strain. The original open reading frame predicted for Nbl1 in the Pombe Genome Database was a truncated version, comprising only the first 95 amino acids of Nbl1's 141 amino acids (Supplementary Figure 1C). Thus, we initially placed nbl1(1-95) cDNA under control of the thiamine repressible nmt41 promoter (Basi et al., 1993) and integrated this fragment at the leu1 locus in a nbl1-disrupted strain. This truncated cDNA was sufficient to rescue the nbl1 disruption, indicating that the C terminus of Nbl1 is dispensable for proper Nbl1 function. Consistent with this, most sequence conservation between S. pombe Nbl1 and homologues in S. octosporus and S. japonicus lies in the N terminus (Supplementary Figure 1C). Because Nbl1(1-95) was fully functional, we used this truncation interchangeably with full-length Nbl1 in our studies. Shutoff of nbl1(1-95) expression produced cut phenotypes (Figure 1C) similar to those which were seen in nbl1-disrupted cells (Figure 1B). Therefore, the previously described defects in chromosome segregation are attributable to disruption of nbl1.
To examine whether nbl1 overexpression is tolerated by cells, nbl1+ cDNA was placed under control of the nmt1 promoter in pREP1 (Maundrell, 1990, 1993) and transformed into wild-type cells. On removal of thiamine, cells overexpressing nbl1 failed to form colonies (Supplementary Figure 1D) and often showed unequal segregation of DNA (Figure 1D). Thus, not only is nbl1 an essential gene, but its protein levels must be regulated properly to ensure appropriate chromosome segregation.
To identify proteins with which Nbl1 interacts and functions, we performed a TAP of Nbl1(1-95) followed by mass spectrometry. Interestingly, Bir1/Cut17, the S. pombe Survivin homologue, and Pic1, the S. pombe INCENP homologue, which both had been previously identified along with Nbl1 in the Clp1-C286S-TAP (Figure 1A), were identified along with Clp1 in a Nbl1(1-95)-TAP (Figure 2A). We confirmed the association between Nbl1(1-95) and Bir1 by traditional coimmunoprecipitation (Figure 2B). In addition to Bir1 and Pic1, Ark1, which is the S. pombe Aurora B homologue, is a known member of the fission yeast chromosomal passenger complex (CPC) (Ruchaud et al., 2007). We did not detect Ark1 in the Nbl1(1-95)-TAP. However, TAPs of the Survivin homologue in S. cerevisiae also failed to recover the Aurora B homologue (Sandall et al., 2006; Widlund et al., 2006), suggesting that the kinase does not remain bound to its nonenzymatic subunits during a standard TAP procedure. Consistent with physical associations between Nbl1 and known CPC components, we also noted a variety of negative genetic interactions among tagged alleles of CPC components and nbl1(1-95). Specifically, nbl1(1-95)-TAP bir1-FLAG, nbl1(1-95)-TAP bir1-GFP, nbl1(1-95)-TAP pic1-myc, and nbl1(1-95)-FLAG bir1-TAP were all synthetically lethal (data not shown).
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; Petersen and Hagan, 2003), and thus histone H3 phosphorylation can be used as a readout for CPC activity. Not surprisingly, histone H3 was phosphorylated by Ark1-GFP from wild-type cells (Figure 2C, lane 3), and such phosphorylation was decreased in the presence of an Aurora B kinase inhibitor, ZM447439 (Figure 2C, lane 2). The specificity of this kinase assay for Ark1 was confirmed using higher concentrations of the Aurora B inhibitor (Supplementary Figure 2). Interestingly, phosphorylation of histone H3 was nearly abolished when Ark1 was immunoprecipitated from cells in which nbl1 expression had been repressed (Figure 2C, lane 4). Although Nbl1 was required for the stability of Ark1 (Figure 2D) in addition to the stability of Bir1 and Pic1 (data not shown), we performed the kinase assay after Ark1 levels had been adjusted for equivalence (Figure 2C, bottom panel). Thus, these results indicate that Nbl1 not only associates with CPC components but is required for proper activity and stability of the CPC.
Nbl1 and the CPC Are Interdependent for Proper Localization
Because Nbl1 and known CPC subunits exhibit physical and functional associations, we next examined whether Nbl1 localizes similarly to chromosomal passenger proteins. As shown by time-lapse microscopy of Nbl1(1-95)-GFP and Sid4-RFP, a marker for the S. pombe spindle pole body (Chang and Gould, 2000), Nbl1(1-95)-GFP localized to dots consistent with centromeres in metaphase and relocated to the mitotic spindle and the spindle midzone during anaphase (Figure 3A and Supplementary Movie 1). This behavior was similar to that of Ark1, Pic1, and Bir1 (Supplementary Figure 3A; Morishita et al., 2001; Petersen et al., 2001; Rajagopalan and Balasubramanian, 2002; Huang et al., 2005). In addition, Nbl1-GFP localized identically to the Nbl1(1-95)-GFP truncation (Supplementary Figure 3B), again demonstrating that Nbl1(1-95) is sufficient for proper localization and function of Nbl1.
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). Colocalization of Nbl1(1-95)-GFP and Nog1-RFP, a nucleolus marker (Matsuyama et al., 2006), confirmed that Nbl1 is also nucleolar in interphase (Figure 3B). Additionally, imaging of Nbl1-GFP with Nuf2-RFP, a kinetochore marker (Nabetani et al., 2001), verified that the Nbl1-GFP dots seen during metaphase were consistent with Nbl1 localization to centromeres in metaphase (Supplementary Figure 3C). Though Nbl1-GFP and Nuf2-RFP did not completely overlap, their side-by-side orientation is similar to that which has previously been observed for other centromeric CPC proteins and kinetochore markers in metaphase (Vanoosthuyse et al., 2007). To further validate that Nbl1 colocalizes with the CPC during mitosis, we imaged Nbl1(1-95)-GFP along with Pic1-mCherry. Nbl1(1-95)-GFP and Pic1-mCherry colocalized to centromeres in metaphase and to the mitotic spindle and the spindle midzone in anaphase (Figure 3C). Therefore, taken together with our evidence for a physical association between Nbl1 and the CPC, it seems likely that Nbl1 travels as a part of the CPC.
We next examined whether Nbl1 requires Bir1, Pic1, or Ark1 for proper mitotic localization. Nbl1-GFP localized normally in wild-type cells at 36°C (Supplementary Figure 3D). At this restrictive temperature in cut17-275 and pic1-T269 temperature-sensitive cells, Nbl1-GFP accumulated at centromeres as usual (16 of 16 cells, 100%; and 38 of 38 cells, 100%, respectively) but commonly failed to localize to the mitotic spindle or the spindle midzone correctly. Instead, Nbl1-GFP often localized diffusely in the nucleoplasm in cut17-275 and pic1-T269 cells (57 of 72 cells, 79%; and 58 of 88 cells, 66%, respectively; Figure 3, D and E), indicating that Bir1 and Pic1 are necessary for its proper spindle localization. Additionally, Nbl1-GFP segregated unevenly at the completion of anaphase (Figure 3, D and E), consistent with unequal segregation of DNA in CPC mutants (Samejima et al., 1993; Leverson et al., 2002). At the restrictive temperature in ark1-T7 temperature-sensitive cells, however, Nbl1-GFP localized normally to both centromeres (53 of 53 cells, 100%) and the mitotic spindle (71 of 74 cells, 96%; Figure 3F). Yet, similar to Nbl1-GFP in cut17-275 and pic1-T269 septated cells, Nbl1-GFP segregated unevenly (Figure 3F). In sum, although Nbl1-GFP localized independently of other CPC components to centromeres, it required the function of Bir1 and Pic1 for proper spindle localization.
Next, we investigated whether localization of known S. pombe CPC subunits requires Nbl1. As previously demonstrated (Morishita et al., 2001; Petersen et al., 2001; Rajagopalan and Balasubramanian, 2002; Huang et al., 2005), Bir1-GFP, Pic1-GFP, and Ark1-GFP localized to centromeres in metaphase and to the mitotic spindle and the spindle midzone in anaphase of wild-type cells (Supplementary Figure 3A). This localization was maintained in nbl1-shutoff cells in the absence of thiamine (Supplementary Figure 3, E–G). However, after repression with thiamine, Bir1-GFP, Pic1-GFP, and Ark1-GFP for the most part did not accumulate at centromeres in metaphase cells (Figure 3, G–I), with 11 of 12 (92%), 15 of 20 (75%), and 19 of 21 (90%) cells lacking clear Bir1-GFP, Pic1-GFP, and Ark1-GFP signals, respectively, at the centromeres. Additionally, during anaphase in nbl1-repressed cells, Bir1-GFP localized diffusely within the nucleoplasm instead of tightly on the spindle in 31 of 35 (89%) cells (Figure 3G). In contrast, Pic1-GFP localized normally on the mitotic spindle and on the spindle midzone in 32 of 36 (89%) cells (Figure 3H). We nonetheless observed unequal segregation after anaphase of both Bir1-GFP and Pic1-GFP (Figure 3, G and H). Because of the high background signal for Ark1-GFP, it was more difficult to ascertain the localization of Ark1-GFP in anaphase upon repression of nbl1 expression. However, we observed separated spindle pole bodies in 52 of 72 (72%) anaphase cells in the absence of any detectable Ark1-GFP signal on the spindle (Figure 3I). This observation suggests that Ark1-GFP localization to the spindle and the midzone, similar to that of Bir1-GFP, was impaired by nbl1 disruption.
Nbl1 Sequence Analysis Reveals Similarities to Human Borealin
Given that Nbl1 associates with known CPC components and colocalizes with them in a dependent manner, it seemed reasonable that Nbl1 might be a fourth subunit of the S. pombe CPC, related to Borealin. To pursue this possibility, we analyzed the predicted secondary structure of Nbl1 using the Jpred3 server (Cole et al., 2008). The N-terminal region of Nbl1, from residues 14 to 85, is predicted to be highly 
-helical (Figure 4A). Additionally, Nbl1 exhibits coiled-coil oligomer potential from residues 22 to 56 of this helical region. Using BLAST to search for related proteins, we identified the helical region of Borealin as a sequence homologue. The central helical region of Nbl1, spanning residues 32 to 79, is 20% identical and 52% homologous to residues 25 to 72 of the N terminus of Borealin (Figure 4B). We independently analyzed Nbl1 using the Phyre protein threading server to search for homologues with known structure (Bennett-Lovsey et al., 2008). The first match was the N terminus of human Borealin, with an E value of 0.35 and estimated precision of 85% (PDB = 2RAX, Bourhis et al., 2007). All other matches were unrelated helical proteins with significantly lower similarity (E value >2.7, precision <55%).
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). The Phyre-based model of Nbl1 (N29-H83) is consistent with core features of Borealin important for this interaction, containing both an extended coiled-coil followed by a helical region similar to the region of Borealin which binds to the dimerization arm of Survivin (Figure 4C). Additionally, the specific residues conserved between Borealin and Nbl1 strongly cluster to those residues at the protein-protein interaction surface. Twenty-two of the 25 conserved residues are predicted to be localized to the protein–protein interaction face of Nbl1 (Figure 4D). The three residues positioned on the opposite helical face (E33, R44, R70) are charged residues. One of the strictly conserved residues is proline 69 (Figure 4, B and D). This proline serves to break the long helix of Borealin and positions the helical segments which bind to the dimerization arm of Survivin (Figure 4D). This interaction leads to the observed dimer to monomer transition of Survivin which is important for Survivin function (Bourhis et al., 2007). Interestingly, although cells were viable when we truncated Nbl1 to its first 95 amino acids, truncation of Nbl1 after the conserved proline at residue 69 rendered cells inviable. Specifically, nbl1(1-69) cells cut in their first division (Figure 4E). Thus, the additional short helical stretch following the conserved proline in Nbl1 appears to be likewise relevant for CPC function in S. pombe. Accordingly, Nbl1 and Borealin share a conserved N-terminal half that is both physically and functionally important. Together with the physical and functional associations of Nbl1 with the CPC, these similarities further support the identification of Nbl1 as the S. pombe Borealin homologue.
Clp1 and the CPC Are also Interdependent for Proper Localization
We next analyzed in more detail the association between CPC components and the Clp1/Cdc14 phosphatase, given that they had purified in our Clp1-C286S-TAP. Phosphatase-dead Clp1-C286S-GFP coimmunoprecipitated Nbl1(1-95)-FLAG during and after release from an nda3-KM311 prometaphase arrest (Figure 5A), suggesting that Clp1 and Nbl1 associate in both metaphase and anaphase. Consistent with this, Clp1-GFP and Nbl1(1-95)-mTomato colocalized to centromeres in metaphase and to the mitotic spindle and the spindle midzone in anaphase (Figure 5B).
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A previous report suggested that Clp1 is required for proper centromere targeting of Ark1 and Bir1 (Trautmann et al., 2004). We thus examined whether localization of Nbl1 is perturbed in clp1
cells. Centromere localization of Nbl1-GFP was intact in all 21 clp1 cells examined (Supplementary Figure 5A). Also, in contrast to the earlier report, we detected all other CPC proteins at centromeres in clp1 cells (n 
20; Supplementary Figure 5A). Thus, we conclude that Clp1 does not affect CPC localization to centromeres. However, though Nbl1-GFP, like the rest of the CPC, initially localized to the mitotic spindle appropriately in clp1 cells (n 20; Supplementary Figure 5B), Nbl1-GFP did not localize to the midzone correctly in clp1 cells (Figure 5C). Instead, it tailed off to one pole during anaphase (Figure 5C). We noted that Nbl1-GFP localized in an identical manner in a strain with the phosphatase-dead clp1-C286S mutation (Supplementary Figure 6, A and B), additionally supporting the conclusion that Clp1 phosphatase activity is relevant to only the midzone localization of the CPC.
Midzone localization of Ark1 in S. pombe has been linked previously to a requirement for Ase1, a microtubule-bundling protein that is necessary for midzone stability (Yamashita et al., 2005). In clp1 (Figure 5D) and clp1-C286S (Supplementary Figure 6C) cells, we observed that Ase1-GFP also tailed off to one pole during anaphase. This suggests that the midzone itself was improperly formed in clp1 and clp1-C286S strains. Thus, mislocalization of Nbl1 and other CPC components in clp1 and clp1-C286S strains appears to be attributable to faulty midzone formation in the absence of Clp1 phosphatase activity.
We next tested whether disruption of nbl1 affects Clp1 localization by monitoring Clp1-GFP and Sid4-RFP after repression of nbl1 expression. In anaphase of wild-type cells, Clp1-GFP localized to both the spindle and the CR (Figure 6E, left column). However, in nbl1-shutoff cells, Clp1-GFP localized to the spindle but not the CR during anaphase in 23 of 33 (70%) cells (Figure 6A). In addition, Clp1-GFP often accumulated between separated spindle pole bodies in an unsegregated mass (Figure 6A), consistent with the chromosome segregation defects seen in nbl1-disrupted cells. We then examined Clp1-GFP and Sid4-RFP in the CPC temperature strains cut17-275, pic1-T269, and ark1-T7. Though some cut17-275, pic1-T269, and ark1-T7 cells showed localization of Clp1-GFP to the CR in early anaphase at the restrictive temperature (panel I, Figure 6, B–D), most anaphase cut17-275 (27 of 35, 77%), pic1-T269 (24 of 31, 67%), and ark1-T7 (36 of 42, 86%) cells lacked any detectable Clp1-GFP signal at the CR (panels III-V, Figure 6, B–D). Instead, Clp1-GFP solely localized to the mitotic spindle and to an unsegregated mass between spindle pole bodies in these cells (panels II-V, Figure 6, B–D). Thus, Clp1-GFP accumulation at the CR was abnormal in all CPC mutations tested.
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The CPC Genetically Interacts with the S. pombe Cytokinetic Apparatus
Clp1 accumulation at the CR is required for the fidelity of cytokinesis (Trautmann and McCollum, 2005; Chen et al., 2008; Clifford et al., 2008). Furthermore, temperature-sensitive alleles of CR components exhibit a strong genetic interaction with mid1431-481, which encodes a Mid1 mutant that lacks the region necessary to recruit Clp1 to the CR (Clifford et al., 2008). Therefore, given the relevance of the CPC to Clp1 accumulation at the CR, we used a genetic approach to examine whether previously unrecognized connections between the S. pombe CPC and the cytokinetic apparatus exist. Interestingly, we observed negative genetic interactions (Figure 7A) between ark1-T7 and temperature-sensitive alleles of the following genes: cdc12, which encodes a critical cytokinetic formin (Pelham and Chang, 2002; Kovar et al., 2003); rng2, which encodes an IQGAP-related protein required for CR formation (Eng et al., 1998); and sid2, which encodes a kinase that functions in septation initiation (Balasubramanian et al., 1998). The fact that the ark1-T7 cdc12–112 double mutant failed to grow as well as either single mutant at the permissive temperature was consistent with the observation that some of these double mutants failed in their first division on tetrad plates (Supplementary Figure 7A). Furthermore, the observed negative genetic interactions between ark1-T7 and CR mutant alleles specifically correlated with an exacerbation of cytokinesis and septation defects, for double mutants showed a greater proportion of defective cells than either of the relevant single mutants (Figure 7, B and C). In addition, we noted that two temperature-sensitive alleles of bir1, cut17-275 and bir1-T1, also showed a negative genetic interaction with cdc12–112 (Supplementary Figure 7, B and C) and that the relevant double mutants likewise exhibited an increased instance of cytokinesis and septation defects (Supplementary Figure 7, D and E). We did not, however, detect genetic interactions between CPC temperature-sensitive alleles and the mid1431-481 mutant allele (Supplementary Figure 8), consistent with the primary role of the CPC in cytokinesis being to mediate Clp1 accumulation at the CR.
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). Though high-dose Lat A treatment (>10 µM) results in a complete loss of F-actin structures in wild-type cells (Pelham and Chang, 2001), low-dose Lat A treatment (0.2 µM) is not lethal (Mishra et al., 2004). However, low-dose Lat A treatment mildly perturbs the cytokinetic machinery, and cells defective in cytokinesis, such as those lacking clp1, are sensitive to these low doses (Figure 8, A–C) (Mishra et al., 2004). Though growth of ark1-T7 on control and low-dose Lat A-containing plates was similar at 25°C, growth of ark1-T7 was slightly impaired at 27°C and considerably impaired at 29°C on low-dose Lat A-containing plates (Figure 8A). Consistent with this observation, ark1-T7 cells showed significant cytokinesis and septation defects in the presence of low-dose Lat A in liquid culture (Figure 8, B and C). Therefore, ark1-T7 cells are sensitive to mild inhibition of actin polymerization at semipermissive temperature. These data support the notion that the fission yeast CPC promotes the process of cytokinesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Vader et al., 2006; Ruchaud et al., 2007), the S. pombe proteins Ark1, Pic1, and Bir1 function within a chromosomal passenger complex that regulates critical mitotic events such as chromosome segregation and spindle elongation (Morishita et al., 2001; Petersen et al., 2001; Leverson et al., 2002; Rajagopalan and Balasubramanian, 2002; Huang et al., 2005; Widlund et al., 2006). However, whether these three proteins operate alone in this complex has remained unclear, for a Borealin homologue has not been previously described in S. pombe. Here, we characterize Nbl1 as an S. pombe Borealin-like protein based on its association with known CPC components, its colocalization with other CPC subunits during mitosis, and its sequence and proposed structural similarity to human Borealin. Consistent with previous data directly linking Borealin to Aurora B activation (Jelluma et al., 2008), we additionally demonstrate that Nbl1 is required for Ark1 activity. Furthermore, we show that the S. pombe CPC influences the process of cytokinesis, at least partially by controlling the localization of the fission yeast Cdc14-family phosphatase, Clp1.
Identification of Borealin homologues in yeasts has previously been hampered by the fact that they do not possess open reading frames with significant primary sequence identity to human Borealin. Even the Nbl1(1-95) sequence, which was sufficient to rescue disruption of nbl1 and to support proper localization and associations of the CPC during mitosis, possesses only a few residues which are identical to residues at the same positions in Borealin. While this manuscript was in preparation, a study was published describing a budding yeast Borealin homologue (Nakajima et al., 2009). In this article, the authors also identified putative Borealin homologues in almost 200 species through Hidden Markov Model–based searches. One of the putative homologues which they noted in this analysis (Nakajima et al., 2009) is the S. pombe protein, which we have described in this study. Thus, our work supports their findings and confirms that Nbl1 is in fact an S. pombe Borealin homologue.
We fortuitously discovered that a truncation of Nbl1 lacking the last 46 amino acids localizes correctly, interacts with other CPC components, and does not exhibit genetic interactions consistent with a loss of function. Though a recent study suggested that full-length Borealin is required for the formation of a competent Borealin–Survivin complex which can bind INCENP (Zhou et al., 2009), it has been posited that Borealin-like proteins in lower eukaryotes retain only structures involved in formation of the critical three-helix bundle of Borealin-Survivin-INCENP homologues (Nakajima et al., 2009). Proper function of Nbl1(1-95) supports this hypothesis because it maintains the core three-helix bundle region. Nbl1(1-95) also retains a second helical stretch that follows a conserved proline. Given that this helical stretch is additionally necessary for viability, this region represents another critical structural domain. It will be interesting to analyze whether this region, similar to the helical stretch following the conserved kink in human Borealin (Bourhis et al., 2007), blocks homodimerization of the Survivin homologue.
A significant question in the CPC field concerns how CPC localization is regulated by its different subunits. In human cells, CPC proteins are strikingly interdependent, with knockdown or disruption of any of the CPC subunits impeding proper localization of the others (Honda et al., 2003; Gassmann et al., 2004). Although mutation of Bir1 has been found to impede localization of Ark1 and Pic1 (Morishita et al., 2001; Rajagopalan and Balasubramanian, 2002; Huang et al., 2005), a comprehensive dissection of CPC interdependency in localization has been lacking in S. pombe. Here, we undertook a thorough analysis of Nbl1-CPC interdependency and noted additional associations relevant to CPC localization. An intriguing possibility suggested by our data is that Nbl1 mediates centromeric targeting of the CPC. Nbl1 localization to centromeres is independent of Bir1, Pic1, and Ark1, whereas centromere localization of Bir1, Pic1, and Ark1 requires Nbl1. Additionally, similar to human Borealin (Klein et al., 2006), Nbl1 binds DNA (unpublished data). Future studies in the genetically tractable S. pombe could help clarify the contribution of Borealin homologues to CPC centromere targeting.
Our data furthermore highlight that Clp1, similar to Cdc14 in S. cerevisiae (Pereira and Schiebel, 2003; Stoepel et al., 2005), is a bonafide CPC-interacting protein. Ark1 has previously been shown to associate with Clp1 during mitosis (Trautmann et al., 2004), and we additionally identified Bir1, Pic1, and Nbl1 in our Clp1-C286S-TAP complexes. The fact that Clp1 and the CPC colocalize during mitosis furthermore confirms their close association. An obvious question stemming from these observations is whether S. pombe CPC proteins are directly regulated by their phosphorylation state. While an in-depth analysis of CPC phosphoregulation in S. pombe is beyond the scope of this study, we have found that Nbl1, similar to human Borealin (Kaur et al., 2007), can be phosphorylated by Cdk1 in vitro and that there are numerous sites of phosphorylation matching the Cdk1 consensus on Bir1 and Pic1. Similar to the recent observation that Survivin function in chicken cells does not require Cdk phosphorylation of a known phosphosite (Yue et al., 2008), mutation of the sole Cdk1 consensus site on Nbl1 did not apparently affect its function or localization. However, there might be a combinatorial effect of CPC subunit phosphorylation, similar to the situation in S. cerevisiae in which redundant Cdk1-mediated phosphorylation blocks DNA rereplication (Nguyen et al., 2001). Thus, an overt phenotype might only be revealed by eliminating most or all Cdk1 phosphorylation of the CPC.
Currently, it is also unclear whether dephosphorylation of S. pombe CPC proteins by Clp1 occurs and, if so, whether this is important for CPC function. The fact that Nbl1, Bir1, and Pic1 copurified with the substrate-trapping Clp1-C286S mutant suggests, however, that at least one of the three is a direct target of Clp1. This possibility is supported by a previous report indicating that Clp1 phosphatase activity is required for Clp1 to affect chromosome segregation through the CPC (Trautmann et al., 2004). Although this issue is unresolved, a few points regarding CPC localization in clp1 cells deserve noting. Though it was suggested that deletion of clp1 affects localization of CPC subunits to centromeres (Trautmann et al., 2004), our data indicate that the CPC localizes normally to centromeres in the absence of Clp1. Also, unlike Cdc14 (Pereira and Schiebel, 2003), Clp1 is not required for the initial spindle recruitment of any CPC component in S. pombe. Therefore, it is most likely that if Clp1 affects the CPC directly, it would influence its localization dynamics or its specific activity. Although Nbl1 localization to the midzone is disrupted in both clp1 and clp1-C286S cells, we have found that the midzone itself is disrupted in the absence of Clp1 activity. In S. cerevisiae, Cdc14 controls midzone formation via de-phosphorylation of Ase1 (Khmelinskii et al., 2007). Our data suggest that Clp1-Ase1 interactions might similarly control midzone assembly in S. pombe.
It had not been examined previously whether the CPC in any organism affects Cdc14 family function. We found, unexpectedly, that Clp1 accumulation at the CR was defective in all tested CPC mutations. Clp1 contributes to CR stability and the fidelity of cytokinesis (Trautmann and McCollum, 2005; Chen et al., 2008; Clifford et al., 2008). Yet, because Clp1 is nonessential in S. pombe, its loss at the CR perturbs, but does not normally prevent, cytokinesis. Consistent with the CPC affecting this aspect of cytokinesis integrity, ark1-T7 cells are sensitive to low-dose Lat A treatment and temperature-sensitive alleles of genes encoding CPC and CR components display negative genetic interactions. Therefore, although Ark1 is not essential for S. pombe cytokinesis (Petersen and Hagan, 2003), our data indicate that Ark1 does play a role in this process.
The CPC has similarly been implicated in S. cerevisiae cytokinesis. In S. cerevisiae, distinct passenger complexes control septin organization in anaphase (Gillis et al., 2005; Thomas and Kaplan, 2007), and the Aurora B homologue mediates a NoCut cytokinesis checkpoint by recruiting abscission inhibitors when DNA fails to segregate out of the cleavage plane (Norden et al., 2006; Mendoza et al., 2009). Though the extent of the contribution of S. pombe CPC to cytokinesis is currently unclear, it is unlikely to monitor DNA remaining in the division plane. Cut mutations resulting from inhibition of a variety of mitotic factors are readily obtained in S. pombe (Yanagida, 1998), indicating that there is no robust mechanism in this organism to delay or prevent cytokinesis when chromosomes remain in the division plane. Furthermore, the genetic interactions we have uncovered indicate that the CPC promotes, rather than inhibits, cytokinesis in S. pombe. In future studies, it will be important to determine whether the sole function of the CPC in S. pombe cytokinesis involves regulating Clp1 localization.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Present address: Department of Cell and Molecular Biology, Grand Valley State University, Allendale, MI 49401.
Address correspondence to: Kathleen L. Gould (kathy.gould{at}vanderbilt.edu).
Abbreviations used: BF, bright field; CPC, chromosomal passenger complex; CR, contractile ring; LatA, latrunculin A.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bahler, J., Wu, J. Q., Longtine, M. S., Shah, N. G., McKenzie, A., 3rd, Steever, A. B., Wach, A., Philippsen, P., and Pringle, J. R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951.[CrossRef][Medline]
Balasubramanian, M. K., McCollum, D., Chang, L., Wong, K. C., Naqvi, N. I., He, X., Sazer, S., and Gould, K. L. (1998). Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 149, 1265–1275.
Basi, G., Schmid, E., and Maundrell, K. (1993). TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123, 131–136.[CrossRef][Medline]
Bennett-Lovsey, R. M., Herbert, A. D., Sternberg, M. J., and Kelley, L. A. (2008). Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70, 611–625.[CrossRef][Medline]
Bourhis, E., Hymowitz, S. G., and Cochran, A. G. (2007). The mitotic regulator Survivin binds as a monomer to its functional interactor Borealin. J. Biol. Chem 282, 35018–35023.
Carmena, M. (2008). Cytokinesis: the final stop for the chromosomal passengers. Biochem. Soc. Trans 36, 367–370.[CrossRef][Medline]
Chang, L., and Gould, K. L. (2000). Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. Proc. Natl. Acad. Sci. USA 97, 5249–5254.
Chen, C. T., Feoktistova, A., Chen, J. S., Shim, Y. S., Clifford, D. M., Gould, K. L., and McCollum, D. (2008). The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1. Curr. Biol 18, 1594–1599.[CrossRef][Medline]
Clifford, D. M., Wolfe, B. A., Roberts-Galbraith, R. H., McDonald, W. H., Yates, J. R., 3rd, and Gould, K. L. (2008). The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1. J. Cell Biol 181, 79–88.
Cole, C., Barber, J. D., and Barton, G. J. (2008). The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36, W197–W201.
Earnshaw, W. C., and Bernat, R. L. (1991). Chromosomal passengers: toward an integrated view of mitosis. Chromosoma 100, 139–146.[CrossRef][Medline]
Eng, K., Naqvi, N. I., Wong, K. C., and Balasubramanian, M. K. (1998). Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr. Biol 8, 611–621.[CrossRef][Medline]
Esteban, V., Blanco, M., Cueille, N., Simanis, V., Moreno, S., and Bueno, A. (2004). A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast. J. Cell Sci 117, 2461–2468.
Gassmann, R., Carvalho, A., Henzing, A. J., Ruchaud, S., Hudson, D. F., Honda, R., Nigg, E. A., Gerloff, D. L., and Earnshaw, W. C. (2004). Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle. J. Cell Biol 166, 179–191.
Gillis, A. N., Thomas, S., Hansen, S. D., and Kaplan, K. B. (2005). A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis. J. Cell Biol 171, 773–784.
Gould, K. L., Moreno, S., Owen, D. J., Sazer, S., and Nurse, P. (1991). Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. EMBO J 10, 3297–3309.[Medline]
Gould, K. L., Ren, L., Feoktistova, A. S., Jennings, J. L., and Link, A. J. (2004). Tandem affinity purification and identification of protein complex components. Methods 33, 239–244.[CrossRef][Medline]
Honda, R., Korner, R., and Nigg, E. A. (2003). Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis. Mol. Biol. Cell 14, 3325–3341.
Huang, H. K., Bailis, J. M., Leverson, J. D., Gomez, E. B., Forsburg, S. L., and Hunter, T. (2005). Suppressors of Bir1p (Survivin) identify roles for the chromosomal passenger protein Pic1p (INCENP) and the replication initiation factor Psf2p in chromosome segregation. Mol. Cell. Biol 25, 9000–9015.
Jelluma, N., Brenkman, A. B., van den Broek, N. J., Cruijsen, C. W., van Osch, M. H., Lens, S. M., Medema, R. H., and Kops, G. J. (2008). Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment. Cell 132, 233–246.[CrossRef][Medline]
Jensen, S., Segal, M., Clarke, D. J., and Reed, S. I. (2001). A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. J. Cell Biol 152, 27–40.
Jeyaprakash, A. A., Klein, U. R., Lindner, D., Ebert, J., Nigg, E. A., and Conti, E. (2007). Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together. Cell 131, 271–285.[CrossRef][Medline]
Kaur, H., Stiff, A. C., Date, D. A., and Taylor, W. R. (2007). Analysis of mitotic phosphorylation of borealin. BMC Cell Biol 8, 5.[CrossRef][Medline]
Keeney, J. B., and Boeke, J. D. (1994). Efficient targeted integration at leu1–32 and ura4–294 in Schizosaccharomyces pombe. Genetics 136, 849–856.[Abstract]
Khmelinskii, A., Lawrence, C., Roostalu, J., and Schiebel, E. (2007). Cdc14-regulated midzone assembly controls anaphase B. J. Cell Biol 177, 981–993.
Klein, U. R., Nigg, E. A., and Gruneberg, U. (2006). Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP. Mol. Biol. Cell 17, 2547–2558.
Kovar, D. R., Kuhn, J. R., Tichy, A. L., and Pollard, T. D. (2003). The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J. Cell Biol 161, 875–887.
Leverson, J. D., Huang, H. K., Forsburg, S. L., and Hunter, T. (2002). The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis. Mol. Biol. Cell 13, 1132–1143.
Mata, J., Lyne, R., Burns, G., and Bahler, J. (2002). The transcriptional program of meiosis and sporulation in fission yeast. Nat. Genet 32, 143–147.[CrossRef][Medline]
Matsuyama, A. et al. (2006). ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol 24, 841–847.[CrossRef][Medline]
Maundrell, K. (1990). nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J. Biol. Chem 265, 10857–10864.
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123, 127–130.[CrossRef][Medline]
Mendoza, M., Norden, C., Durrer, K., Rauter, H., Uhlmann, F., and Barral, Y. (2009). A mechanism for chromosome segregation sensing by the NoCut checkpoint. Nat. Cell Biol 11, 477–483.[CrossRef][Medline]
Mishra, M., Karagiannis, J., Trautmann, S., Wang, H., McCollum, D., and Balasubramanian, M. K. (2004). The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe. J. Cell Sci 117, 3897–3910.
Morishita, J., Matsusaka, T., Goshima, G., Nakamura, T., Tatebe, H., and Yanagida, M. (2001). Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair. Genes Cells 6, 743–763.[Abstract]
Nabetani, A., Koujin, T., Tsutsumi, C., Haraguchi, T., and Hiraoka, Y. (2001). A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint. Chromosoma 110, 322–334.[CrossRef][Medline]
Nakajima, Y., Tyers, R. G., Wong, C. C., Yates, J. R., 3rd, Drubin, D. G., and Barnes, G. (2009). Nbl1p: A Borealin/Dasra/CSC-1-like protein essential for Aurora/Ipl1 complex function and integrity in Saccharomyces cerevisiae. Mol. Biol. Cell 20, 1772–1784.
Nguyen, V. Q., Co, C., and Li, J. J. (2001). Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411, 1068–1073.[CrossRef][Medline]
Norden, C., Mendoza, M., Dobbelaere, J., Kotwaliwale, C. V., Biggins, S., and Barral, Y. (2006). The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage. Cell 125, 85–98.[CrossRef][Medline]
Ohi, R., Sapra, T., Howard, J., and Mitchison, T. J. (2004). Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. Mol. Biol. Cell 15, 2895–2906.
Pelham, R. J., and Chang, F. (2002). Actin dynamics in the contractile ring during cytokinesis in fission yeast. Nature 419, 82–86.[CrossRef][Medline]
Pelham, R. J., Jr, and Chang, F. (2001). Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe. Nat. Cell Biol 3, 235–244.[CrossRef][Medline]
Pereira, G., and Schiebel, E. (2003). Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14. Science 302, 2120–2124.
Petersen, J., and Hagan, I. M. (2003). S. pombe aurora kinase/survivin is required for chromosome condensation and the spindle checkpoint attachment response. Curr. Biol 13, 590–597.[CrossRef][Medline]
Petersen, J., Paris, J., Willer, M., Philippe, M., and Hagan, I. M. (2001). The S. pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation. J. Cell Sci 114, 4371–4384.[Medline]
Pines, J., and Rieder, C. L. (2001). Re-staging mitosis: a contemporary view of mitotic progression. Nat. Cell Biol 3, E3–6.[CrossRef][Medline]
Queralt, E., and Uhlmann, F. (2008). Cdk-counteracting phosphatases unlock mitotic exit. Curr. Opin. Cell Biol 20, 661–668.[CrossRef][Medline]
Rajagopalan, S., and Balasubramanian, M. K. (2002). Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis. Genetics 160, 445–456.
Roberts-Galbraith, R. H., Chen, J. S., Wang, J., and Gould, K. L. (2009). The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring. J. Cell Biol 184, 113–127.
Ruchaud, S., Carmena, M., and Earnshaw, W. C. (2007). Chromosomal passengers: conducting cell division. Nat. Rev. Mol. Cell Biol 8, 798–812.[CrossRef][Medline]
Samejima, I., Matsumoto, T., Nakaseko, Y., Beach, D., and Yanagida, M. (1993). Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis. J. Cell Sci 105, (Pt 1), 135–143.[Abstract]
Sandall, S., Severin, F., McLeod, I. X., Yates, J. R., 3rd, Oegema, K., Hyman, A., and Desai, A. (2006). A Bir1-Sli15 complex connects centromeres to microtubules and is required to sense kinetochore tension. Cell 127, 1179–1191.[CrossRef][Medline]
Steigemann, P., Wurzenberger, C., Schmitz, M. H., Held, M., Guizetti, J., Maar, S., and Gerlich, D. W. (2009). Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484.[CrossRef][Medline]
Stoepel, J., Ottey, M. A., Kurischko, C., Hieter, P., and Luca, F. C. (2005). The mitotic exit network Mob1p-Dbf2p kinase complex localizes to the nucleus and regulates passenger protein localization. Mol. Biol. Cell 16, 5465–5479.
Thomas, S., and Kaplan, K. B. (2007). A Bir1p Sli15p kinetochore passenger complex regulates septin organization during anaphase. Mol. Biol. Cell 18, 3820–3834.
Trautmann, S., and McCollum, D. (2005). Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation. Curr. Biol 15, 1384–1389.[CrossRef][Medline]
Trautmann, S., Rajagopalan, S., and McCollum, D. (2004). The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase. Dev. Cell 7, 755–762.[CrossRef][Medline]
Vader, G., Medema, R. H., and Lens, S. M. (2006). The chromosomal passenger complex: guiding Aurora-B through mitosis. J. Cell Biol 173, 833–837.
Vagnarelli, P., and Earnshaw, W. C. (2004). Chromosomal passengers: the four-dimensional regulation of mitotic events. Chromosoma 113, 211–222.[CrossRef][Medline]
Vanoosthuyse, V., Prykhozhij, S., and Hardwick, K. G. (2007). Shugoshin 2 regulates localization of the chromosomal passenger proteins in fission yeast mitosis. Mol. Biol. Cell 18, 1657–1669.
Widlund, P. O., Lyssand, J. S., Anderson, S., Niessen, S., Yates, J. R., 3rd, and Davis, T. N. (2006). Phosphorylation of the chromosomal passenger protein Bir1 is required for localization of Ndc10 to the spindle during anaphase and full spindle elongation. Mol. Biol. Cell 17, 1065–1074.
Wolfe, B. A., and Gould, K. L. (2004). Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation. EMBO J 23, 919–929.[CrossRef][Medline]
Wolfe, B. A., McDonald, W. H., Yates, J. R., 3rd, and Gould, K. L. (2006). Phospho-regulation of the Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe. Dev. Cell 11, 423–430.[CrossRef][Medline]
Yamashita, A., Sato, M., Fujita, A., Yamamoto, M., and Toda, T. (2005). The roles of fission yeast ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling. Mol. Biol. Cell 16, 1378–1395.
Yanagida, M. (1998). Fission yeast cut mutations revisited: control of anaphase. Trends Cell Biol 8, 144–149.[CrossRef][Medline]
Yoon, H. J., Feoktistova, A., Wolfe, B. A., Jennings, J. L., Link, A. J., and Gould, K. L. (2002). Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes. Curr. Biol 12, 2048–2054.[CrossRef][Medline]
Yue, Z. et al. (2008). Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line. J. Cell Biol 183, 279–296.
Zeng, X., Kahana, J. A., Silver, P. A., Morphew, M. K., McIntosh, J. R., Fitch, I. T., Carbon, J., and Saunders, W. S. (1999). Slk19p is a centromere protein that functions to stabilize mitotic spindles. J. Cell Biol 146, 415–425.
Zhou, L., Li, J., George, R., Ruchaud, S., Zhou, H. G., Ladbury, J. E., Earnshaw, W. C., and Yuan, X. (2009). Effects of full-length borealin on the composition and protein-protein interaction activity of a binary chromosomal passenger complex. Biochemistry 48, 1156–1161.[CrossRef][Medline]
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