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Vol. 18, Issue 6, 2047-2056, June 2007
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*Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309
Submitted October 13, 2006;
Revised March 12, 2006;
Accepted March 19, 2007
Monitoring Editor: Tim Stearns
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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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; Cleveland et al., 2003; Taylor et al., 2004). It helps ensure that all sister chromatids are bioriented, with sisters attached to opposite spindle poles, before anaphase onset. This checkpoint is not essential in yeasts; indeed, deletion of the mitotic arrest defective (MAD) genes leads to a rather subtle phenotype in the absence of additional perturbation of the mitotic machinery (Warren et al., 2002). However, mouse knockouts of Mad2 and Bub3 are early embryonic lethal (Dobles et al., 2000; Kalitsis et al., 2000), and it has been argued that this is because the spindle checkpoint plays a critical role in the timing of mitosis in vertebrates (Meraldi et al., 2004). Efficient RNA interference (RNAi) of Mad and Bub components in tissue culture shortens mitosis so much that it is catastrophic, because there simply is not enough time to build a spindle and attach all kinetochores properly before anaphase onset (Meraldi et al., 2004; Michel et al., 2004).
The nonessential nature of the yeast MAD genes makes them extremely tractable genetically, and allowed us to perform a synthetic lethal screen with the aim of identifying novel mitotic defects that activate the spindle checkpoint. Neither the mitotic mutation nor a mad1
on their own kill cells, but when combined they become lethal. Synthetic lethal screens with mad mutants have been carried out using the viable haploid gene deletion set, and many interactions found, including a range of nonessential spindle and kinetochore functions (Lee and Spencer, 2004; Daniel et al., 2006). Our screen is unbiased: any essential or nonessential component of the mitotic machinery that can be mutated to cause a defect that is recognized by the spindle checkpoint can be identified. A pilot screen that identified 25 mitotic mutants was conducted (Hardwick, unpublished data), but among them were four novel alleles of sfi1.
SFI1 was initially identified as a suppressor of the fil1 mutation (Ma et al., 1999), and it has since been demonstrated to be an essential and widely conserved component of centrosomes, which are known in yeast as spindle pole bodies (SPBs) (Kilmartin, 2003).
The budding yeast spindle pole body is a layered structure embedded in the nuclear envelope and is the sole microtubule-organizing center, nucleating both nuclear and cytoplasmic microtubules from its inner and outer plaques, respectively. Associated with one side of the SPB is a structure called the half-bridge, a specialization of nuclear membrane with a layer of material on the cytoplasmic face (O'Toole et al., 1999). The half-bridge serves several important roles during spindle pole body duplication. On initiation of SPB duplication, the half-bridge extends and a small satellite body is formed on the distal cytoplasmic tip (Byers, 1981). The satellite expands to form a duplication plaque, which is then inserted in the membrane by an unknown process, possibly involving contraction of the half-bridge (Adams and Kilmartin, 1999). After the inner plaque assembles on the nuclear face of duplication plaque, the result is two side-by-side SPBs joined by the bridge. The bridge then cleaves or dissociates, allowing the two SPBs to separate and organize opposite ends of the spindle.
One important component of the half-bridge is Cdc31p, the single budding yeast member of the conserved centrin family. Members of the centrin family have important roles in microtubule-organizing centers such as the SPB, centrosome, and basal bodies; and in higher organisms, they are often the major component of Ca2+-sensitive contractile fibers (Schiebel and Bornens, 1995). Sfi1p biochemically interacts with Cdc31p, is localized to the half-bridge of the SPB, and like Cdc31p is required for early steps in SPB duplication (Kilmartin, 2003). Sfi1p contains a central domain composed of multiple, evolutionarily conserved repeats, each of which can bind to Cdc31p (Kilmartin, 2003). It was suggested that Sfi1p–centrin fibers could act as elastic connections between various elements of the centrosome and that they might mediate a licensing step necessary for centrosome duplication (Salisbury, 2004). Recently, the crystal structures of two Sfi1p–centrin complexes were reported, and it was confirmed that multiple molecules of Cdc31p bind a single Sfi1p, resulting in a filamentous structure (Li et al., 2006). On the basis of electron microscopy (EM) evidence placing the N terminus of Sfi1p at the SPB and the C terminus at the center of the bridge, it was proposed that an Sfi1p/Cdc31p filament could span the length of the half-bridge. Kilmartin and coworkers suggested that during SPB duplication, the half-bridge could double in length through association of two, end-to-end Sfi1 C termini, providing a new Sfi1 N terminus as an assembly site for the new SPB (Li et al., 2006). However, these important functions for the N- and C-terminal domains of Sfi1p have yet to be investigated experimentally.
Here, we describe new sfi1 mutants that arrest in mitosis with duplicated, side-by-side SPBs, indicating that Sfi1p does have further roles to play in addition to its previously described function in centrin-mediated SPB duplication. These mutations all map to the C terminus of Sfi1p, and they are entirely consistent with models in which the Sfi1p C terminus has an important role in separation or splitting of the duplicated SPBs during bipolar spindle assembly.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Standard stocks were 10 mg/ml 
factor, 10 mg/ml nocodazole, 100 mg/ml hydroxyurea, and 30 mg/ml benomyl. Yeast transformation and polymerase chain reaction (PCR)-mediated epitope tagging were carried out using standard procedures (Ito et al., 1983; Longtine et al., 1998), and for mCherry (Shaner et al., 2004; Snaith et al., 2005). DNA sequencing was carried out by PCR according to manufacturer's instructions (ABI Big Dye; Applied Biosystems, Foster City, CA). Table 1 lists the yeast strains used, all of which are derivatives of W303.
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).
Linkage Analysis of the Novel sfi1 Alleles
To test genetically whether the synthetic lethal mutations were tightly linked to the sfi1 locus, we constructed a wild-type SFI1, mad1 strain in which the URA3 marker was integrated just upstream of SFI1. This strain was then crossed with the synthetic lethal mutants, diploids were sporulated, and tetrads were dissected. If the synthetic lethal mutation was linked to the SFI1 locus, no spores would be recovered that were URA3+ and that contained the synthetic lethal mutation. The latter was assessed visually, using the MAD1, ADE3 plasmid and colony sectoring assay. We dissected at least 24 tetrads for each sfi1 allele, and we never saw recombination between the mutation and URA3. This showed that each mutation was tightly linked to, and most likely lay within, the SFI1 gene. This was later confirmed by sequencing these sfi1 alleles (Figure 1B).
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and bub1
Sfi1p Antibodies
Nucleotides 1-1655 of SFI1 were PCR amplified and cloned into pGEX-6P. Bacterial cultures were induced with isopropyl 
-D-thiogalactoside overnight at 18°C, pelleted, frozen, and then ground in a pestle and mortar under liquid nitrogen. Extracts were thawed in lysis buffer (phosphate-buffered saline, 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Tween 20), sonicated for 1 min, and clarified. Sfi1–glutathione S-transferase (GST) fusion protein was then purified on glutathione agarose by using standard protocols, dialyzed overnight (50 mM HEPES, pH 7.6, 100 mM KCl, and 30% glycerol), and used as antigen. Anti-Sfi1p serum was affinity purified on a column of Sfi1-GST coupled to Affigel 10 (Bio-Rad, Hercules, CA) as described previously (Hardwick and Murray, 1995). Immunoblotting was carried out as described previously (Hardwick and Murray, 1995).
Light Microscopy
Strains were grown in complete synthetic media (CSM) or the appropriate selective CSM media (Qbiogene, Carlsbad, CA) made up to double recommended concentration. For GFP/YFP/CFP/mCherry imaging, cells were analyzed live, or they fixed briefly with formaldehyde (typically 3.7% for 5–10 min). Microscopy was performed with a Zeiss Axiovert 200 Marianas inverted microscope system from Intelligent Imaging Innovations (Denver, CO), with a CoolSNAP HQ charge-coupled device camera. Images were taken and analyzed using Slidebook software (Intelligent Imaging Innovations).
Electron Microscopy
Cells were fixed, processed, and imaged as described previously (Giddings et al., 2001). Twenty-four sfi1 cells were analyzed very thoroughly by serial section.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mutants. These strains are red if they maintain the ADE3 plasmid, but they are white if they lose it. The synthetic lethal mutants die if they lose the MAD1, ADE3 plasmid and therefore grow to form entirely red colonies (Figure 1A). These four mutants were all rescued with an SFI1 clone from a YCP50-based genomic library (Hardwick and Murray, 1995), enabling them to lose the MAD1, ADE3 plasmid and form sectored colonies (data not shown, but see Figure 6). Linkage experiments suggested that the synthetic lethal mutations were likely to be sfi1 alleles rather than being suppressed by the SFI1 gene (see Materials and Methods for details). The sfi1 locus was then PCR amplified using genomic DNA from all four strains, sequenced, and found to contain mutations in, or very close to, the C-terminal domain of Sfi1p (Figure 1B). This domain lies after the central repeat domain that has been shown to interact with Cdc31p. The sfi1-273 allele (R789K) actually lies in the last of the 21 repeats thought to interact with centrin (Li et al., 2006), but it could perturb the overall structure of the C terminus. Although the C terminus is not conserved in other model organisms or human Sfi1p, database analysis showed that it, if the last repeat is included, is the section of Sfi1p with the highest levels of homology among budding yeast (ranging from 40% identity for Saccharomyces cerevisiae and Saccharomyces castelli to 98% identity for S. cerevisiae and S. paradoxus; Figure 1C). This suggested that the C terminus of Sfi1p has an important conserved function, at least in budding yeast.
Their isolation as mad1 synthetic lethal mutations suggested that these sfi1 alleles have mitotic defects, even at 23°C, because they are dependent on the spindle checkpoint for viability. To further our understanding of their phenotypes, we analyzed the growth of these sfi1 alleles in different conditions, and we compared them with the tight temperature-sensitive sfi1-3 allele (this contains mutations H207Q, F208C, Y247C; Kilmartin, 2003). The new sfi1 mutants grew significantly slower than wild type at 18 and 36°C, although their cold-sensitive phenotype was somewhat variable. We analyzed their temperature sensitivity further by shifting log phase cultures to 36°C for 4 h and then analyzing cells by fluorescence-activated cell sorting and microscopy. No clear DNA replication defect was observed by FACS (Figure 2B), but a significant increase in the number of large-budded cells was observed in all four strains (Figure 2C). Such an accumulation of large-budded cells with 2C DNA is consistent with a mitotic delay due to spindle checkpoint activation. Next, we tested whether these sfi1 alleles were sensitive to antimicrotubule drugs, and we found them to be benomyl resistant (Figure 2D). This is a relatively unusual phenotype and suggests the possibility that these mutants somehow stabilize microtubules, thereby counteracting the effects of antimicrotubule drugs.
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and bub1 (see Materials and Methods for details), indicating that this is not a genetic interaction that is specific to the loss of Mad1p function.
Protein Stability and Localization of Sfi1p Are Unaffected
Next, we analyzed the mutant Sfi1 proteins, to determine whether their stability was affected upon temperature shift. To do this, we raised polyclonal antibodies to the N terminus of Sfi1p (see Materials and Methods), and we used these on immunoblots of total protein extracts. These antibodies recognized wild-type Sfi1p, which ran at the predicted size of 112 kDa. As expected, a truncated protein was detected in sfi1-229 (97 kDa), and a larger fusion protein of 
140 kDa was recognized in the strain expressing Sfi1-YFP (Figure 2F). We immunoblotted extracts made from wild-type and sfi1 mutant cells grown at 23 and 37°C. This showed that all of the mutant Sfi1 proteins were stable, even at 37°C (Figure 2G). We conclude that the C-terminal sfi1 mutations do not significantly affect the stability of the Sfi1 protein.
We used double-label fluorescence experiments to confirm that Sfi1p localizes to budding yeast SPBs (Figure 3A; Kilmartin, 2003). To determine whether our mad1 synthetic lethal sfi1 alleles were defective in SPB targeting, we fused the monomeric red fluorescent protein mCherry (Shaner et al., 2004) to sfi1-120. To ensure identical exposure times and image processing, we imaged a mixture of mCherry-tagged sfi1-120p mutant cells, which also expressed Spc42-green fluorescent protein (GFP), and mCherry-tagged wild-type Sfi1p cells that did not express Spc42-GFP. The sfi1-120 mutant protein was still detected at SPBs (Figure 3B), and quantitative analysis showed that it was there at wild-type levels (Figure 3C). Thus, the sfi1 C-terminal point mutants do not seem to affect the stability of Sfi1p or its targeting to the SPB.
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). Analysis of spindles in sfi1-65 and sfi1-120 alleles revealed a very distinct phenotype (Figure 4). We presynchronized cells in G1 with -factor, and then we released them to 37°C for 3 h, before fixing them briefly with formaldehyde. When analyzed by light microscopy, using cyan fluorescent protein (CFP)-tubulin and Spc42-GFP, we observed a significant enrichment of cells with paired GFP spots (Figure 4, A and B). The two Spc42-GFP spots were typically 0.3 µm apart. These spindle poles were much closer together than in a normal short mitotic spindle (1.2 µm in early wild-type mitosis), and they had a very similar separation to the spindles observed in cdc4 mutants (also 0.3 µm), which are known to arrest with duplicated but unseparated SPBs (Figure 4C; Byers and Goetsch, 1974). This suggested that the new sfi1 alleles had arrested with side-by-side SPBs or with very short bipolar spindles.
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-factor arrested and then released at 37°C for 3 h before high-pressure freezing. Although some large-budded cells had bipolar spindles that looked normal, 50% had duplicated side-by-side SPBs (Figure 5). These SPBs were of equal and normal size, had a clear half-bridge connecting them, and had nuclear microtubules emanating from them. This demonstrates that the sfi1-65 and sfi1-120 defect is postduplication and that it does not affect the ability of the SPBs to nucleate microtubules. Thus, these sfi1 mutants have a defect in SPB separation. This analysis by electron microscopy demonstrates that Sfi1p has an additional role to play in bipolar spindle formation after SPB duplication.
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). To confirm that the sfi1 C-terminal defect(s) of mutants was mechanistically distinct from that of sfi1-3 and sfi1-7, we tested whether CDC31 overexpression could also suppress their temperature sensitivity. This was not the case (Figure 6A). We next tested a bank of high copy (2-µm) plasmids encoding other SPB components to see whether any of them could suppress the sfi1 C-terminal alleles. To do this, we tested whether their overexpression could suppress the synthetic lethality of the sfi1 C-terminal mutant alleles with mad1, by using the MAD1, ADE3 plasmid and colony sectoring assay. These sfi1 mutants require MAD1 for viability, and by maintaining the MAD1, ADE3 plasmid, they form red colonies (because they are chromosomally ade2, ade3 mutants). If the 2-µm plasmid suppresses the sfi1 mutation, they are then able to lose the ADE3 plasmid and form red/white sectored colonies (Figure 6B). As expected, plasmids encoding either SFI1 or MAD1 allowed frequent colony sectoring. In addition, we found that BBP1, NDC1, MPS2, KAR1, and CIN8 plasmids could all suppress the synthetic lethality to different extents (Figure 6C). Bbp1, Mps2, and Ndc1 are all implicated in the process of inserting the newly formed SPB duplication plaque into the nuclear membrane, and they possibly have roles in anchoring the SPB once inserted (Chial et al., 1998; Schramm et al., 2000; Park et al., 2004; Araki et al., 2006). This suggests that at least part of the sfi1 C-terminal mutant phenotype could reflect a defective interaction between the SPB and the nuclear envelope. However, our EM analysis does not support an insertion defect, because the duplicated SPBs seem to be sitting normally in the nuclear envelope, and they are able to nucleate microtubules. It is not known what other roles the Mps2/Bbp1 membrane protein complex has to play in spindle biogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple Functions for Sfi1p
Sfi1p had already been shown to have a role in the initial stage of SPB duplication. The sfi1-3 and sfi1-7 alleles, which have mutations in the Cdc31p binding repeats, arrest with only one spindle pole body before satellite assembly (Kilmartin, 2003). Recent work from the Kilmartin laboratory has described the crystal structure of Sfi1p–Cdc31p complexes (Li et al., 2006). In addition, it was shown by EM shadowing that Sfi1p–Cdc31p complexes form filaments in isolation. The authors proposed an elegant model describing the role of Sfi1p in SPB duplication. The model has Sfi1p molecules spanning the half-bridge with their N termini anchored in the spindle pole body and their C termini at the distal end. They suggested that upon initiation of SPB duplication additional Sfi1p molecules are recruited to the half-bridge and interact with the existing half-bridge Sfi1p molecules, end on, via their C termini. This end-on interaction results in a doubling of the bridge length, and the Sfi1 C termini are now positioned at the center of the bridge (Figure 7). This model was strongly supported by immuno-EM analysis of strains that expressed Sfi1p with either N- or C-terminal GFP tags: the N terminus was positioned next to the junction between the SPB and the proximal end of the half-bridge, whereas the C-terminal tag was found to be close to the distal end, or in the center of the bridge in paired SPBs (Li et al., 2006). Thus, the Sfi1p C termini are ideally placed to mediate interactions necessary for bridge separation. It was suggested that an important role for the Sfi1p C terminus might be in mediating the cleavage process, either alone or in combination with other proteins.
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Our genetic studies confirm that the sfi1 C-terminal mutant phenotype is novel and shows that it is not due to a defective interaction with Cdc31p, as was found for the sfi1-3 and sfi1-7 alleles. In addition, our multicopy suppression analysis identified other components of the spindle pole body that can partially rescue the defect of sfi1 C-terminal mutations. One of these components was the kinesin motor protein Cin8p. This is a plus-end–directed motor required to separate duplicated SPBs by producing an outward force on the bipolar spindle (Saunders and Hoyt, 1992; Saunders et al., 1997). We suggest that overexpression of the Cin8 motor protein might increase this outwards force to such an extent that it is capable of splitting and driving apart the duplicated SPBs in the sfi1-229 and sfi1-273 mutants.
Some of the sfi1 suppressors encode integral membrane proteins (Ndc1p, Mps2p) of the nuclear envelope, and others are known to form a complex with them (Mps2-Bbp1-Nbp1). The terminal phenotype of conditional alleles of these SPB components is typically a "dead" SPB, which fails to nucleate nuclear microtubules (Chial et al., 1998; Schramm et al., 2000; Park et al., 2004; Araki et al., 2006). This is due to a failure to insert the new SPB into the nuclear envelope. Could these complexes interact with the Sfi1p C termini? It was recently shown that bridge components, such as Sfi1p, Cdc31p, Mps3p, and Kar1p, are all present on these dead SPBs (Araki et al., 2006), so it is unlikely that these factors are involved in targeting or maintaining Sfi1p at the bridge. Whether the "insertion" factors have later roles to play in spindle biogenesis is unclear, although it is thought that Nbp1 is required to maintain Mps2p at the SPB (Araki et al., 2006). We propose that the C termini of Sfi1p do have an important role to play in bridge splitting, as proposed originally (Li et al., 2006), and that this separation event might require interaction with integral components of the nuclear envelope, such as the Mps2–Bbp1 complex (see model in Figure 7).
Almost nothing is known about separation of the SPB bridge. Another mutant that arrests at a similar stage is cdc4, an F-box component of the ubiquitin ligase known as the SCF (Mathias et al., 1996; Cardozo and Pagano, 2004). This suggests the possibility that an SCF substrate needs to be degraded, by ubiquitin-mediated proteolysis, for bridge separation to occur. It is tempting to think that a half-bridge linker molecule is itself the SCF substrate, but it is also possible that the SCF substrate regulates bridge separation. Indeed, SCF activity may simply be needed to get to the correct stage of the cell cycle for SPB separation to occur, perhaps through the activation of microtubule motors and changes in microtubule dynamics after S phase onset. That increased Cin8 motor protein expression can suppress two of the sfi1 alleles is interesting and suggests that increased motor activity can be sufficient to drive defective Sfi1p C termini apart leading to SPB separation.
It will be of interest to discover whether Sfi1 homologues have roles to play in splitting and separation of vertebrate centrosomes, and if so, whether the Sfi1 proteins are perturbed in cancers. Many human tumors display centrosome aberrations, but it is unclear as to whether these are typically the cause or consequence of cancer progression (Nigg, 2002). Either way, it is possible that the spindle checkpoint could make a useful drug target for anticancer therapy, because we have shown here that the combined loss of Sfi1p and Mad1p functions can be completely lethal to cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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Medical Research Council Radiation and Genome Stability Unit, Harwell, Oxon, United Kingdom; 
Department of Energy Joint Genome Institute, 2800 Mitchell Dr., Walnut Creek, CA 94598.
Address correspondence to: Kevin G. Hardwick (kevin.hardwick{at}ed.ac.uk).
Abbreviations used: EM, electron microscopy; MAD, mitotic arrest defective; NAT, nourseothricin; SPB, spindle pole body.
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