Correspondence to Kenneth B. Kaplan: kbkaplan{at}ucdavis.edu
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Introduction |
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CBF3 consists of multiple subunits; the Ctf13p subunit forms the core of the complex, physically contacting both Cep3p and Ndc10p and, together, making direct contacts with CEN DNA (Espelin et al., 1997; Kaplan et al., 1997; Russell et al., 1999; Rodrigo-Brenni et al., 2004). Assembly of this complex is necessary for binding CEN DNA and requires Skp1p, Sgt1p, and the HSP90 chaperone (Kitagawa et al., 1999; Stemmann et al., 2002; Bansal et al., 2004; Lingelbach and Kaplan, 2004). The assembly of CBF3 is balanced by its turnover; Ctf13p is targeted for ubiquitin-mediated degradation and CBF3 complexes are themselves unstable (Lingelbach and Kaplan, 2004; Rodrigo-Brenni et al., 2004). We refer to this balance of assembly and turnover as the CBF3 cycle, and we have shown that this cycle occurs throughout the cell division cycle. Inhibiting CBF3 assembly does not disrupt kinetochores on CEN DNA, neither by GFP fusion localization nor by chromatin immunoprecipitation (Rodrigo-Brenni et al., 2004). Our interpretation of these results is that CBF3 complexes are stably bound to CEN DNA and that the CBF3 cycle may contribute to a noncentromeric role for CBF3.
Although kinetochore assembly on CEN DNA leads to the formation of a microtubule attachment site, the dynamic properties of kinetochores have been associated with mitotic progression. For example, spindle checkpoint proteins associate with kinetochores early in mitosis and are down-regulated after chromosomes have formed proper bivalent attachments with the mitotic spindle (Cleveland et al., 2003; Lew and Burke, 2003). In anaphase, the coordination of chromosome segregation with the start of cytokinesis is associated with the transfer of passenger proteins from kinetochores to the central spindle (Vagnarelli and Earnshaw, 2004; Yang et al., 2004).
The association of passenger complexes with kinetochores has been proposed to allow the proper position of these proteins on the central spindle during anaphase. However, a wealth of evidence now indicates that these proteins also play an important role in chromosome congression and in resolving misoriented kinetochore spindle attachments (Petersen et al., 2001; Tanaka et al., 2002; Lampson et al., 2004). How these early mitotic roles relate to the function of passenger proteins in cytokinesis is unclear. Inhibition studies have shown that the passenger proteins Aurora B, INCENP (inner centromere protein), and Survivin are required for cytokinesis in animal cells (Terada et al., 1998; Kaitna et al., 2000; Terada, 2001; Murata-Hori et al., 2002; Yang et al., 2004), but it is not known exactly how these proteins regulate the cytokinetic apparatus. In yeast, it is unknown if the passenger protein homologues Ipl1p, Sli15p, and Bir1p also have a role in cytokinesis. Recent studies have shown that Ipl1p and Sli15p localize to interpolar microtubules and regulate spindle stability in anaphase (Buvelot et al., 2003; Pereira and Schiebel, 2003). Interestingly, the CBF3 mutant ndc10-1 similarly affects spindle stability and compromises cytokinesis, suggesting that kinetochore complexes may participate in regulating multiple anaphase events (Ducat and Zheng, 2004; Bouck and Bloom, 2005).
We show that the kinetochore scaffold complex CBF3 has an unexpected second role in regulating septins and cytokinesis. Several lines of evidence suggest that this second role is independent of its function in forming kinetochores and segregating chromosomes: (a) only CBF3 mutants, but not mutants in outer kinetochore complexes, affect septin organization; (b) SKP1 and SGT1 alleles that affect CBF3 turnover, but not chromosome segregation, compromise septin organization; and (c) inhibiting CBF3 in cells arrested in G1 with mating pheremones also causes defects in septin organization. Measurements of septin dynamics in anaphase reveal that the CBF3 cycle is required for proper ring separation and disassembly of the mother septin ring; these defects contribute to aberrant polarized cell growth, bud site selection, and cytokinesis. Mutants in the passenger protein homologue BIR1, but not in IPL1 or SLI15, also result in septin organization defects. These results lead us to propose that CBF3 and Bir1p function as a passenger complex that regulates septins and cytokinesis.
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Results |
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Disorganized septins may reflect a failure to properly regulate their dynamics during the cell cycle. In addition, we observed the most severe septin defects in anaphase cells (Fig. S1 D). To examine the requirement for CBF3 assembly during anaphase more directly, we arrested strains that contain Cdc11-GFP and either CTF13 or GAL1-CTF13 in S phase, using hydroxyurea. Cells were transferred to media containing raffinose for 2 h in the presence of hydroxyurea (Fig. 2 A). We observed no change in septin organization after the hydroxyurea arrest in either control or GAL1-CTF13 strains (Fig. 2 B, +raffinose HU arrest). Cells were released from S phase by washing out the hydroxyurea and returning them to medium containing raffinose with or without nocodazole, a microtubule poison that arrests cells in metaphase. In cells arrested in metaphase, inhibition of CBF3 assembly had no effect on the Cdc11-GFP pattern (Fig. 2 B, +raffinose, HU release, +nocodazole). Finally, we released cells from the hydroxyurea-arrest into medium containing raffinose and allowed them to progress past metaphase; fixing and staining cells indicated that >90% of large budded cells were in anaphase under these conditions (unpublished data). In control cells, we observed the normal separation of septin rings marking the beginning of anaphase. In contrast, a high percentage of cells inhibited for CBF3 assembly exhibited disorganized septins during this same time period (Fig. 2, B [+raffinose, HU release] and C). From these data, we concluded that CBF3 assembly is critical for septin organization after the movement of chromosomes to the poles in anaphase, at a time when septins are separating to form the cytokinetic furrow.
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Septins are used in yeast throughout the cell cycle to control bud growth as well as cytokinesis. To examine whether the defects in septin organization compromise cytokinesis, we monitored the GAL1-CTF13 strain when grown over longer periods of time in either galactose or raffinose. When CBF3 assembly was inhibited (raffinose), we observed an increase in the number of cells with segregated DNA masses and extended anaphase spindles, which is consistent with a defect in cytokinesis (Fig. 6 A). In previous studies, it has been shown that compromised cytokinesis can give rise to multinucleated cells because of defects in septins when the CDK/cyclin regulator SWE1 is deleted (Sreenivasan and Kellogg, 1999). We examined the appearance of multinucleated anaphase cells in a GAL1-CTF13, SWE1 strain. We observed a small percentage of multinucleated cells when CBF3 assembly was inhibited in a wild-type SWE1 strain and a dramatic increase in multinucleated cells when SWE1 was deleted (952% of anaphase cells; Fig. 6, B and C). The accumulation of anaphase cells with aberrant septins and the increase in multinucleated cells strongly argue that CBF3 assembly is critical for cytokinesis.
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The requirement of CBF3 to regulate septins during anaphase is reminiscent of the role ascribed to passenger proteins in higher eukaryotes. Interestingly, we and others have observed that CBF3 subunits are found both at kinetochores and on interpolar microtubules during anaphase (Fig. S3, Ndc10-GFP), a localization change that is similar to passenger protein behavior in animal cells (Buvelot et al., 2003; Pereira and Schiebel, 2003). Furthermore, in yeast BIR1 (Survivin), IPL1 (Aurora B) and SLI15 (INCENP) encode likely homologues of known passenger proteins, and both Ipl1p and Sli15p have been shown to localize to interpolar microtubules late in anaphase (Buvelot et al., 2003; Pereira and Schiebel, 2003). We have confirmed these localizations (not depicted), and we now show that Bir1-GFP also localizes to kinetochores early in the cell cycle and is found on interpolar microtubules in anaphase (Fig. 7 A). Previous work identified that the CBF3 subunit, Ndc10p, interacts with Bir1p via yeast two hybrid (Yoon and Carbon, 1999). We confirmed this result and showed that neither Ipl1p nor Sli15p interact with Ndc10p by yeast two hybrid (Fig. 7 B). To show that the interaction between Ndc10p and Bir1p is direct, we translated Ndc10p in vitro and incubated it with GST or a GST-Bir1p fusion. The enrichment of translated Bir1p with GST-Bir1p but not GST is consistent with the direct interaction between these two proteins (Fig. 7 C), indicating that CBF3 and Bir1p may function together.
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Discussion |
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Although it is surprising that a sequence-specific DNA-binding complex has a second role in regulating septins, our previous work suggested that the dynamic assembly and turnover of CBF3 continues after kinetochores have formed on CEN DNA; this observation led us to propose that the CBF3 cycle may be required for processes independent of its role in nucleating kinetochores and segregating chromosomes (Rodrigo-Brenni et al., 2004). We show that septin organization is sensitive to perturbations in the CBF3 cycle; mutations that inhibit CBF3 assembly (skp1-4) or the depletion of CBF3 from cells (raffinose/GAL1-CTF13) cause defects in the organization of septins. Blocking the turnover of CBF3 complexes (skp1-3) or overproducing CBF3 (galactose/GAL1-CTF13) also compromises septin organization. Although the precise role of the CBF3 cycle in regulating septins remains unclear, we speculate that this cycle may maintain the balance of CBF3 complexes that form kinetochore scaffolds versus passenger complexes.
The role of septins in cytokinesis has been well established in multiple systems (Kinoshita, 2003; Longtine and Bi, 2003). Recently, in yeast, septins have been shown to form a diffusion barrier in anaphase, which helps to ensure the proper localization of the membrane and cell wall synthesis machinery required for cytokinesis (Dobbelaere and Barral, 2004). Even subtle perturbations in septin organization lead to diffusion of these proteins away from the mother bud neck and failed cytokinesis (Dobbelaere and Barral, 2004). Certainly, the septin defects that we observed are consistent with the loss of this diffusion barrier activity and may explain the defect in cytokinesis. Septin organization may be compromised because of a failure to properly regulate their dynamics late in anaphase. Although our data support a role for CBF3 in regulating these dynamics, we imagine that this role is indirect, possibly through changes in posttranslational modifications of septins. The regulation of septin dynamics during the anaphaseG1 transition has been linked to protein phosphorylation, as well as SUMO (small ubiquitin-like modifier) modification (Johnson and Blobel, 1999; Johnson and Gupta, 2001; Shih et al., 2002; Kinoshita, 2003; Martin and Konopka, 2004). It has been reported that the PP2A subunit Rts1p localizes to kinetochores in metaphase and to septins in anaphase (Dobbelaere et al., 2003). Our comparison of the septin phenotypes suggests that RTS1 and CBF3 differentially regulate septins. Nonetheless, it is striking that defects in all of these pathways (i.e., phosphorylation, sumoylation, and ubiquitination) affect aspects of septin stability. It is possible that CBF3 intersects with multiple regulatory pathways that control septin separation and disassembly, thus creating a more complex phenotype than individual mutations in septin regulators.
Our observation that inhibiting CBF3 assembly also affects septins in factortreated cells has multiple implications. We conclude that CBF3 assembly is required in G1 to regulate septins, completely independent of its role in segregating chromosomes. The belief that septins are probably dynamic as they reform from a shmoo into bud configurations supports a role for CBF3 in regulating septin dynamics. Therefore, this result may reflect a requirement of CBF3 to regulate septins when they are at their most dynamic; e.g., during the disassembly of septin rings at the anaphaseG1 transition. Furthermore, this finding may reflect a similar need in anaphase and mating cells to coordinate microtubules, actin, and nuclear movement. In this light, it is intriguing to note that, during mating in yeast microtubules, nucleate from opposite spindle poles must find each other with the help of the septinactin cortex and form a plus-endplus-end interaction. This orientation of microtubules is analogous to the orientation of anaphase interpolar microtubules and suggests that these two structures may share some of the same machinery to form their attachment sites.
The direct interaction between CBF3 and Bir1p indicates that these proteins function together to regulate septins in anaphase. The importance of this interaction, and the possibility that other proteins participate in regulating septins, remains to be explored. Our genetic data suggest that neither Sli15p nor Ipl1p are involved in regulating septins (Fig. 7). This is somewhat surprising, as Survivin, which is the Bir1p homologue in animal cells, interacts with Aurora B (Ipl1p) and INCENP (Sli15p), and Bir1p has been observed to copurify with Sli15p from yeast (Wheatley et al., 2001; Cheeseman et al., 2002). Although we have examined multiple alleles of IPL1 and SLI15, it is possible that these mutations remain functional for regulating septins. ipl1-321 has been reported to eliminate the kinase activity of Ipl1p (Biggins et al., 1999), showing that the kinase function of Ipl1p is not critical for septin regulation. Caution is also required when interpreting the alleles of SLI15, especially because sli15-1 was isolated as a synthetic lethal mutant of ipl1-2 and sli15-3 displays a very similar phenotype to ipl1-2 (Kim et al., 1999); it may well be that these SLI15 alleles are specifically defective in regulating Ipl1p kinase activity but wild type for its role in a putative septin-regulating complex. Nonetheless, the high frequency of conditional BIR1 alleles that we have isolated with septin defects demonstrates that Bir1p has a role distinct from the Sli15pIpl1p complex in regulating septin dynamics (unpublished data). Studies in animal cells are consistent with this possibility, as Survivin is found in multiple passenger-type complexes (Gassmann et al., 2004). Whether the CBF3Bir1p passenger complex directly interacts with septins or whether it influences intermediates to regulate septins is an important question that remains to be addressed.
Are these novel findings regarding passenger complexes in yeast relevant to higher eukaryotes? Although CBF3 is not conserved, there are some very intriguing similarities between budding yeast and higher eukaryote passenger complexes. For example, Sgt1p, Skp1p, and HSP90 regulate the CBF3 cycle and are highly conserved (Kitagawa et al., 1999; Bansal et al., 2004; Lingelbach and Kaplan, 2004; Rodrigo-Brenni et al., 2004). It has been shown that the small interfering RNA of human SGT1 compromises kinetochore formation, supporting the argument that a similar cycle may impact human kinetochores (Steensgaard et al., 2004). In higher eukaryotes, CENP-Acontaining nucleosomes are critical for kinetochore formation. Like CBF3, CENP-A is a target of the Aurora B passenger kinase (Ipl1p in yeast; Sassoon et al., 1999; Zeitlin et al., 2001). Remarkably, overexpression of a CENP-A mutant that prevents Aurora B phosphorylation results in cytokinetic defects (Zeitlin et al., 2001). These results raise the interesting possibility that kinetochore scaffold complexes share a conserved dual role in nucleating kinetochores and in coordinating chromosome segregation with cytokinesis. Finally, the transfer of Bir1p from kinetochores to interpolar microtubules and its requirement for cytokinesis is reminiscent of Survivin, its counterpart in mammalian cells (Yang et al., 2004). Therefore, we conclude that an analogous system of proteins function in yeast to coordinate chromosome segregation with septin organization and cytokinesis.
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Materials and methods |
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In vitro binding assay
Recombinant GST and GST fused to Bir1p were produced in insect cells using the Fastbac Baculovirus expression system (Invitrogen). Proteins were isolated after cell lysis, as previously described (Rodrigo-Brenni et al., 2004). Ndc10p was translated in vitro using the TNT T7 Quick Coupled Translation/Transcription System (Promega). 0.5 µg of template DNA and 20 µCi of 35S-methionine were used in the translation reaction, as per the manufacturer's recommendations. GlutathioneSepharose (GE Healthcare) was used to isolate 1 µg GST-Bir1p or GST and mixed with half of the translation reaction in 250 µL of binding buffer (50 mM Tris, pH 7.0, 50 mM KCl, 0.05% Triton X-100, 1 mM DTT, and 10% glycerol). Samples were rocked at 4°C for 3 h, the resin was washed three times with binding buffer, and proteins were resolved using SDS-PAGE and analyzed using Phosphorimager analysis (GE Healthcare).
Cell cycle point of execution
In these experiments, strains contained either the wild-type CTF13 or the GAL1 promoter integrated upstream of the CTF13 coding sequences and fused to a triple HA epitope tag, as previously reported (Rodrigo-Brenni et al., 2004). Cells were grown at 30°C to log phase in YEP media plus 2% galactose and 2% raffinose. The medium was adjusted to 0.1 M hydroxyurea, and >90% of cells arrested with large buds over a 3-h period. Cells were centrifuged and resuspended in hydroxyurea containing YEP with 2% raffinose or 2% raffinose/galactose, as indicated in the figure legends, and incubated at 34°C for 2 h. Cells were released from the block by centrifugation and washing with fresh medium containing the carbon source indicated in the figure legends and were allowed to proceed through anaphase or arrested during metaphase with 15 µg/ml nocodazole. Cells were collected after 90 min and analyzed by fluorescent microscopy. Abnormal septins were scored based on Cdc11-GFP distribution; all observations were confirmed using immunofluorescence with anti-Cdc11 antibodies.
Cells were similarly arrested using 10 µg/ml factor (American Peptide, Inc.) in medium containing 2% raffinose/galactose. Cells were centrifuged and washed into fresh medium containing
factor and 2% raffinose or 2% raffinose/galactose for 2 h. Finally, cells were centrifuged and washed into fresh medium containing the carbon source indicated in the figure legends and 0.1 M hydroxyurea. Samples were collected after 120 min and analyzed by fluorescent microscopy.
Fluorescent and light microscopy
Cells containing GFP gene fusions were collected at the indicated time points in the figure legends and placed on agarose pads containing a carbon source identical to that of the culture media, as described previously (Hoepfner et al., 2000; Rodrigo-Brenni et al., 2004). For fluorescent staining, cells were grown as indicated in the figure legends, incubated in 3.7% formaldehyde for 1 h at the culture temperature, and processed for DAPI, tubulin, and Cdc11p staining (tubulin or Cdc11p antibodies were diluted 1:400 in PBS, 0.2% gelatin, and 0.02% NaN3) as described previously (Guthrie and Fink, 1991; Rodrigo-Brenni et al., 2004). Phalloidin staining was performed by resuspending the cells in 0.1 M Tris/HCl, pH 9.4, and 10 mM DTT and incubating at 25°C for 8 min. Cells were washed twice with ice-cold PBS; 1.0 x 107 cells were resuspended in 100 µL PBS and supplemented with 4.0 x 103 units of Texas redconjugated phalloidin (Sigma Aldrich) and 2.5 µg DAPI. Cells were incubated in the dark for 1.5 h, washed twice in PBS, resuspended in 20 µL PBS, and applied to poly-L-lysinecoated coverslips. To visualize bud scars, cells were resuspended in PBS with 20 µg/ml Calcofluor white (Fluorescent Brightener 28; Sigma Aldrich), incubated at 25°C for 5 min, washed in PBS, and visualized as described above for GFP fusions.
Fluorescent images were collected using an epifluorescence microscope (model E600; Nikon) equipped with either 60x (NA 1.35) or 100x (NA 1.4) oil immersion lenses (Nikon) and recorded with a charge-coupled device camera (model Orca ER; Hamamatsu) controlled by Simple PCI software (Compix Inc.). Color was added to images using Adobe Photoshop version 7.0. The rat tubulin antibody was purchased from Sigma-Aldrich. The polyclonal antibody against Cdc11p was provided by D. Kellogg (University of California, Santa Cruz, Santa Cruz, CA; Carroll et al., 1998).
Online supplemental material
Fig. S1 shows changes in the kinetics of disorganized septins and abnormal bud morphologies after inhibition of CBF3 complexes. Fig. S2 depicts examples of septin organization in strains containing mutations in inner or outer kinetochore genes. Fig. S3 provides an analysis of septin organization in wild-type and mutant strains of RTS1 and suggests that RTS1 and CBF3 are in parallel pathways with respect to septin regulation. Fig. S4 presents septin organization in additional bir1, sli15, and ipl1 conditional alleles. Video 1 shows the behavior of Cdc11-GFP in anaphase of wild-type cells. Video 2 shows the behavior of Cdc11-GFP in an anaphase inhibited for CBF3 assembly. Video 3 shows an example of septin behavior in an unbudded cell inhibited for CBF3 assembly. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200507017/DC1.
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Acknowledgments |
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We acknowledge the support of a grant from the American Cancer Society (RSG-02-035-01-CCG) to K.B. Kaplan.
Submitted: 6 July 2005
Accepted: 24 October 2005
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References |
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