Correspondence to M.P. Rout: rout{at}mail.rockefeller.edu or C. Strambio-de-Castillia: strambc{at}mail.rockefeller.edu
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J. Fasolo's present address is Yale University, New Haven, CT 06520.
Abbreviations used: IMO, intranuclear microtubule organizer; MTOC, microtubule organizer center; NE, nuclear envelope; NPC, nuclear pore complex; PrA, protein A; SPB, spindle pole body; TEM, transmission electron microscopy.
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Introduction |
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The yeast proteins Mlp1p and Mlp2p belong to a well-conserved family of NE proteins, which in vertebrates is represented by the protooncogenic protein Tpr (Kuznetsov et al., 2002). The Mlps are large, coiled-coil filamentous proteins projecting from the nucleoplasmic side of the NPC (Strambio-de-Castillia et al., 1999; Kosova et al., 2000) and they are nonessential in budding yeast. The Mlps have been proposed to form a peripheral nuclear network whose function is still not clear (Strambio-de-Castillia et al., 1999). Consistent with its localization to the NPC, some data indicate a role for the Mlps in nucleocytoplasmic transport (Strambio-de-Castillia et al., 1999; Kosova et al., 2000). Recent observations also indicate that Mlp1p has a role in RNA biogenesis and in particular in nuclear retention of unspliced mRNAs (Galy et al., 2004; Vinciguerra et al., 2005). The Mlps have also been proposed to be telomeric anchoring sites and involved in the establishment of silent chromatin (Galy et al., 2000; Feuerbach et al., 2002; subsequent studies have called these roles into question, although they did indicate that the Mlps might be implicated in telomere maintenance [Andrulis et al., 2002; Hediger et al., 2002a,b]).
In this study we performed a biochemical analysis, which revealed that Mlp2p binds directly to the SPB core. In the absence of Mlp2p, SPB components are not efficiently targeted to the nuclear face of the SPB, leading to smaller SPBs and to delays and errors of cell division. These results show that the NE is an integrated unit whose structures function coordinately to perform essential nuclear functions.
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Results |
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Mlp2p binds to intact SPB cores
To test if the interaction between Mlp2p and the SPB is limited to unassembled subcomplexes of the SPB or to certain stages during the spindle assembly cycle, we visualized the complex by transmission electron microscopy (TEM). Thin sections of beads from the Mlp1pPrA immunoprecipitation (Fig. 1 c) and empty control beads (unpublished data) were devoid of any visible protein structures. However, the magnetic beads on which the Mlp2pPrA complex was immobilized were covered with numerous discrete structures, 80 nm in diameter (Fig. 1 d, small arrows), which, at higher magnification, resembled isolated SPB cores (Fig. 1 e).
To confirm that Mlp2p-associated structures were indeed bona fide SPB cores, we also compared the isolated structures from either haploid or diploid cells (Fig. 1 f), as diploid SPBs are characteristically larger than haploid SPBs in diameter but not in thickness, and the layered morphology of diploid SPBs is more easily discernible than that of haploid SPBs (Byers and Goetsch, 1975; Bullitt et al., 1997). The structures on the beads isolated from diploid cells were revealed to be morphologically identical to chemically extracted diploid SPB cores (Adams and Kilmartin, 1999). The layered appearance of SPBs was clearly visible in these structures, with the electron-dense central plaque flanked on either side by filamentous protein layers. Structures resembling the bridge, which connects recently duplicated SPBs during early S phase, were also visible in some examples, accounting for the presence of Cdc31p in the Mlp2pSPB complex (Fig. 1 a; Spang et al., 1993). Although the average width of the diploid layered structures was significantly larger than that of the haploid structures, their average thicknesses were the same; all measurements are consistent with previously published values for SPBs. Together, these data establish that the structures associated with Mlp2p are SPB-derived cores.
Core components of the SPB coimmunoprecipitate Mlp2p, but not Mlp1p
To test the specificity of the Mlp2pSPB interaction, we performed reciprocal coimmunoprecipitation experiments followed by immunoblotting. We used Spc42pPrA or Spc110pPrA as baits and Mlp1pMyc or Mlp2pMyc as targets (Fig. 2, coexpressed). We found that Mlp2pMyc was indeed coimmunoprecipitated by both Spc42pPrA and Spc110pPrA, and Mlp1pMyc did not interact with either of the baits. This confirms the high specificity of the Mlp2pSPB interaction, because Mlp1p (a protein similar in location, size and secondary structure to Mlp2p) failed to interact either with Spc42p or Spc110p. We note that Cnm67p-PrA failed to immunoprecipitate either Mlp1pMyc or Mlp2pMyc, in agreement with the in vitro binding experiments (unpublished data).
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Mlp proteins localize to the same nuclear hemisphere as the SPB
With their C-shaped distribution around the rim of the nucleus, the Mlps define a specific hemisphere of the NE, which only partially overlaps with the even distribution of NPCs around the rim of the nucleus and is excluded from the region juxtaposed to the nucleolus (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200504140/DC1; Strambio-de-Castillia et al., 1999; Kosova et al., 2000; Galy et al., 2004). First, to directly compare the localization of the Mlp proteins with respect to each other, we used CFPMlp1p and YFPMlp2p to visualize their position within the same cell (Fig. 3 a). Though the Mlp proteins occupied the same general area of the nuclear periphery, CFPMlp1p was distributed fairly evenly along a C-shaped portion of the nuclear periphery; YFPMlp2p was concentrated into fewer foci covering less area.
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mlp2 cells display significant delays in proceeding past metaphase
To explore the functional significance of the interaction between the SPB and Mlp2p, we determined the position and morphology of the spindle in asynchronous haploid cells by following GFP-tagged Tub1p. In this test, 52% of cells lacking Mlp2p showed a short spindle, characteristic of cells in late S phase or metaphase, compared with 38% for wild-type cells and 39% for mlp1 cells. This result represents a 38% increase of cells in metaphase for mlp2
versus wild type (Fig. 4 a), consistent with Mlp2p having a role in the efficient progression of cells past G2 or early M phase. To assess whether Mlp2p could have a role in promoting spindle migration to the bud neck, we measured the spindle migration index in late S to early M cells (Fig. 4 b). We observed no significant difference between wild-type, mlp1
, and mlp2
cells in this assay. These results suggest that Mlp2p is not involved in the regulation of the activity of cytoplasmic microtubules, consistent with its interaction with SPB components localized only to the nuclear face of the central plaque. To further investigate the function of Mlp2p, we examined wild-type and mlp mutant cells expressing Spc42pGFP. Cells lacking Mlp2p showed an excess number of cells displaying SPB-"doublets" (i.e., cells with two SPBs per cell body) as compared with wild-type and mlp1
cells, consistent with the results we obtained with GFPTub1p-expressing cells (Fig. 4 c). To assess whether Mlp2p might have a role in promoting the formation of a fully formed metaphase spindle, we measured the distance between duplicated SPBs in G2 to early M cells (Fig. 4 d). We found that the average SPB-to-SPB distance was significantly reduced in cells lacking Mlp2p with respect to wild type (i.e., 1.58 µm for wild-type cells; 1.10 µm for mlp2
cells). Taken together, these results points to a role for Mlp2p in facilitating SPB separation and in promoting the formation of a complete metaphase spindle, but not in astral microtubule-directed nuclear migration to the bud neck.
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To demonstrate that the abnormal electron-dense structures we observed by thin-section TEM are indeed amorphous intranuclear assemblies of un-incorporated SPB components, we performed a post-embedding labeling immuno-EM experiment on enriched defective haploid cells carrying the mlp2 allele and expressing Spc42GFP (Fig. 5 d). Gold particles were specifically found in association either with fully formed SPBs at the NE (Fig. 5 d, top left inset, arrowhead) or with intranuclear amorphous structures resembling the ones shown in Fig. 5 c (Fig. 5 d, arrows). This result underscores the specificity of the labeling and demonstrates that the intranuclear electron-dense bodies found in cells lacking Mlp2p contain Spc42p moieties, which failed to be incorporated in the central plaque of fully formed SPBs. Overall, the ultrastrucutural data are consistent with the results we obtained by fluorescent microscopy and suggest that Mlp2p is involved in the proper incorporation of SPB components into fully formed SPBs.
MLP2 interacts genetically with a mutant affecting SPB assembly
To further investigate the functional interactions between the SPB and Mlp2p, we tested temperature-sensitive mutations of various SPB components for genetic interactions with MLP1 and MLP2. When a strain containing the spc110-220 mutation was mated with the mlp2 strain we were unable to recover spores carrying both mutations, although we could generate double mutant strains from all other crosses tested (see Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200504140/DC1). The spc110-220 strain carries a C911R mutation in the Cmd1p-binding domain of Spc110p, which reduces the ability of Spc110p to bind Cmd1p at the restrictive temperature and impairs the incorporation of Spc110p into the central plaque of the SPB, leading to slow growth and the formation of intranuclear mitotic organizers (Sundberg et al., 1996). To study this genetic interaction, we created spc110
strains covered by plasmids expressing either SPC110 or spc110-C911R (thereafter referred to as spc110-220). In addition, we introduced a repressible promoter upstream of either a PrA-tagged MLP1 or MLP2 gene. When grown in dextrose this promoter repressed expression to levels below the immunoblotting detection limit (Fig. S3 available at http://www.jcb.org/cgi/content/full/jcb.200504140/DC1).
Although the SPC110 control strains exhibited equal viability at all temperatures tested, the strains carrying spc110-220 had inhibited growth at 37°C (Fig. 6 a). The additional depletion of Mlp2p from spc110-220 cells led to them being inviable at this temperature. Even at permissive temperatures the depletion of Mlp2p decreased the viability of spc110-220 strains significantly. The repression of MLP1 also reduced viability in conjunction with spc110-220, but to a lesser extent than MLP2. We found that the depletion of MLP2 led to a nearly fourfold increase in aberrant nuclear morphology in the spc110-220 mutant at the restrictive temperature (Fig. 6 b). The nuclei appeared enlarged or fragmented with multiple intense DAPI-stained areas distributed throughout the cell, in contrast to the discrete, round wild-type nuclei. This morphology of the nucleus was reminiscent of the one we observed in multicellular chains present in mlp2 strains (Fig. 5 b). Other described defects associated with spc110-220 at the restrictive temperature were found to be independent of the expression level of MLP2 (unpublished data). We compared the effects of Mlp depletion in the spc110-220 background with the effect of MLP deletion in other temperature-sensitive SPB protein mutants (Fig. S4 available at http://www.jcb.org/cgi/content/full/jcb.200504140/DC1; Fig. 6 c; Geiser et al., 1991; Davis, 1992; Sundberg and Davis, 1997; Elliott et al., 1999). We find that the other mutants we tested showed either mild or no synthetic lethal interactions with MLP2. These results confirm that a specific functional interaction exists between spc110-220, a mutant in a central plaque component affecting SPB assembly, and the Mlps via Mlp2p.
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Discussion |
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Several lines of evidence suggest that one reason for the connection of SPBs to the peripheral Mlp assembly is to aid the incorporation of new components into the nuclear face of the SPB (Fig. 8). First, new SPBs are inserted normally into the NE in cells lacking Mlp2p, suggesting that the protein has no role in the initial stages of SPB assembly. Second, the nucleus migrates normally, a process associated with the cytoplasmically nucleated SPB microtubules, likely excluding Mlp2p from roles associated with the cytoplasmic face of the SPB. Third, Mlp2p is exclusively nuclear, and attaches to the SPB on its nuclear face exclusively via nucleoplasmically oriented SPB components, suggesting that Mlp2p is intimately involved in the function of the SPB, but only after it has inserted into the NE. Fourth, mlp2 cells have an increased failure rate in SPB separation and in progressing past early mitosis, indicating that the nuclear face of SPBs is occasionally compromised in forming proper spindles. Fifth, a significant fraction of the mlp2
population accumulates aberrant intranuclear microtubule organizers, indicating a failure in the proper targeting of components to the SPB. This is similar to the phenotype of the spc110-220 mutant, which also fails to properly target SPB components. Here the defect is in the COOH terminus of the Spc110p protein, where it tightly interdigitates with the Spc42p crystalline layer (Kilmartin et al., 1993; Adams and Kilmartin, 1999). The synthetic lethality we observe in cells lacking Mlp2p and carrying the spc110-220 allele suggests that Mlp2p and the COOH terminus of Spc110p act synergistically in the maturation and maintenance of the SPB. Sixth, SPBs in the mlp2
mutant are smaller on average in comparison to SPBs of a wild-type strain, as indicated by relative SPB fluorescence intensity. Because the thickness of the individual SPB layers is constant, this reduction in fluorescence intensity represents roughly a 10% decrease in surface area. Even though this appears to be only a minor reduction, the surface area of the SPB in yeast limits the number of microtubules emanating from it. A haploid wild-type SPB can nucleate the 16 required kinetochore microtubules and approximately 2 to 4 pole-to-pole microtubules (O'Toole et al., 1999). Because we neither observe chromosome loss nor increased lethality with spindle checkpoint mutants in the mlp2
strain (unpublished data), it appears that capturing of the kinetochores is unimpaired. Thus, the 10% reduction in SPB size might lead to a greater than 50% reduction in the number of pole-to-pole microtubules, consistent with an increase in the number of cells with duplicated but not completely separated SPBs in the mlp2
strain. The requirement for Mlp2p in SPB function is not absolute, as mlp2
cells are viable. Yet, a significant number of mlp2
cells fail to execute mitosis normally. Because loss of Mlp2p can lead to suboptimal SPBs, each new round of SPB duplication might cause the introduction of additional defects due to the dynamic nature of the SPB during S phase. This in turn would lead to stochastic failures of individual SPBs at different stages of the cell cycle. Equally diverse mitotic defects are observed with other (more penetrant) mutants involved in SPB assembly, such as cmd1-1 and SPC110 COOH-terminal mutants (Stirling et al., 1996; Stirling and Stark, 2000).
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Our work also shows that Mlp1p and Mlp2p have overlapping but different distributions at the nuclear periphery, and are functionally distinct. So far, yeasts are the only eukaryotes for which two different Mlps have been identified in the same organism (Kuznetsov et al., 2002). Although in Saccharomyces cerevisiae and its close relatives the second copy arose because of a whole-genome duplication event (Kellis et al., 2004), detailed sequence analyses reveal that in fission yeast this second copy of the Mlp protein arose independently (Ding et al., 2000). Alm1p/TC80, the Schizosaccharomyces pombe MLP2 paralog, localizes to the nuclear rim and appears to accumulate at the SPB and at the medial region MTOC (Ding et al., 2000). Because both budding and fission yeast undergo closed mitosis, with the NE remaining intact throughout the cell cycle, it is conceivable that this kind of direct communication between the NE and spindle organizer may facilitate closed mitosis. Alternatively, it may be necessary for any large structure associated with the NE (be it NPC or SPB) to be anchored into the Mlp assembly. Even though many eukaryotes dispense with their NE during mitosis, the distinction between open and closed mitosis appears to be less than absolute. Thus, the MTOC is linked with the NE during nuclear migration and positioning, and varying amounts of NE stay in the vicinity of the MTOCs during spindle assembly, even in cells with an open mitosis (Nadezhdina et al., 1979; Beaudouin et al., 2002; Malone et al., 2003). Although there is no direct evidence that Mlp/Tpr homologues in vertebrates might play a role similar to that of yeast Mlp2p, in Drosophila it appears that the Tpr homologue Megator aids the formation of a spindle matrix, supporting this idea (Qi et al., 2004). Together with our findings, such results suggest that the close relationship between the NE, Mlp/Tpr, and the MTOC might be a universal feature of the cell division process.
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Materials and methods |
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pRS314-DsRed-Nop1 (Gadal et al., 2001) and pXYNup49-CFP were used in strains expressing either Mlp1pYFP or YFPMlp2p. For pXYNup49-CFP, the enhanced-CFP (eCFP) open reading frame was PCR amplified from pECFP-N1 (BD Biosciences) and inserted into the HindIII and SalI sites of pYX242 (Novagen) to produce pYX242-CFP. The NUP49 open reading frame was PCR amplified from the pET28b-NUP49 plasmid (gift from S. Dokudovskaya, The Rockefeller University, New York, NY) and inserted into the unique BamHI and HindIII sites of pXY242-CFP to produce pXYNup49-CFP. In all cases the plasmid sequence was verified by DNA sequencing.
Affinity PrA purification
The protocol for the purification of PrA-containing complexes was modified from published methods (Aitchison et al., 1996; Schultz et al., 1997). In brief, frozen cells were ground with a motorized grinder (Retsch) and 1 g (PrA control, Mlp1pPrA) or 2 g (Mlp2pPrA) were thawed into 9 ml of extraction buffer 1 (EB1; 20 mM Na-Hepes, pH 7.4, 0.5% Triton X-100, 1 mM DTT, 4 µg/ml pepstatin, 0.2 mg/ml PMSF) supplemented with 300 mM NaCl. Different amounts of frozen cell powder had to be used in order to recover comparable amounts of Mlp-PrA. Cell lysates were homogenized with a Polytron for 25 s (PT 10/35; Brinkman Instruments) and cleared by centrifugation at 2,000 gav for 10 min. 7.5 mg of epoxy-activated Dynabeads (Dynal) cross-linked to rabbit IgG (ICN Biomedicals) were added to each lysate and rotated for 2 h at 4°C. The IgG-Dynabeads were collected with a magnet, washed five times with 1 ml of EB1 and once with 1 ml of 100 mM ammonium acetate, pH 7.4, 0.1 mM MgCl2. The PrA-containing complexes were eluted off the beads in 1 ml of 0.5 M NH4OH, 0.5 mM EDTA at 25°C for 20 min and lyophilized in a SpeedVac (Thermo Savant). Protein samples were resolved by SDS-PAGE with Novex 420% Tris-glycine polyacrylamide gels (Invitrogen) and visualized by Coomassie blue staining.
For the preparation of purified Mlp1p- and Mlp2pPrA for use as probes in the in vitro blot binding assay, the procedure described above was modified as follows. The ground cell powder (1.2 g for Mlp1pPrA and 4 g for Mlp2pPrA) was resuspended in 10 ml of extraction buffer 2 (EB2; 20 mM Hepes/KOH, pH 7.4, 1% Triton X-100, 0.5% Na-deoxycholate, 0.3% sodium N-lauroyl-sarcosine, 0.1 mM MgCl2, 1 mM DTT, 4 µg/ml pepstatin, 0.2 mg/ml PMSF) per gram. After homogenization and clarification, the lysate was incubated over night at 4°C with IgG-Sepharose resin (10 µl of bed volume per gram of cell powder). After extensive washing in EB2 without DTT, bound material was eluted off the resin using a biotinylated derivative of a previously published PrA-mimicking peptide (Biotin-DCAWHLGELVWCT; DeLano et al., 2000). The eluting peptide was removed on a G25 sizing column (Amersham Biosciences) and the probe was quantified by running an aliquot on SDS-PAGE alongside BSA standards.
Identification of proteins by mass spectrometry
Protein bands were excised and tryptic digestions were prepared according to standard protocols (Krutchinsky et al., 2001). Extracted peptides were analyzed by a modified matrix-assisted laser desorption/ionization ion trap mass spectrometer (Krutchinsky et al., 2001) based on a LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan). An automated protocol was used to perform tandem mass spectrometry and proteins were identified searching the National Center for Biotechnology Information nonredundant protein database with the program Sonar (Genomic Solutions) to identify proteins from the tryptic peptide fragmentation masses (Field et al., 2002).
In vitro blot binding assay
The Mlp2p complex was immobilized on IgG-Dynabeads as described for affinity PrA purification. After binding, Dynabeads were incubated using 1 M MgCl2, 20 mM Hepes/KOH, pH 7.4, 0.5% Tween 20 in order to preserve the binding of Mlp2pPrA to IgG and selectively elute the Mlp2pPrA bound proteins off the beads. The eluate was subsequently incubated with fresh IgG-Dynabeads to remove contaminating Mlp2pPrA and with PrA-Sepharose beads to remove IgG that might have bled through from the IgG-Dynabeads. Proteins were then recovered by TCA precipitation, separated on SDS-PAGE, and transferred to nitrocellulose. After amido black staining the blot was cut into three 0.2-cm wide identical strips. The strips were blocked for 1 h at 25°C in 5% milk, 2% BSA, 20 mM Hepes/KOH, 110 mM KOAc, 2 mM MgCl2, 0.1% Tween 20, 1 mM DTT, 4 µg/ml pepstatin, 0.2 mg/ml PMSF, before incubation with 1.2 µg of purified Mlp probes in 500 µl of the same buffer at 4°C overnight. After four brief washes with the same buffer at 25°C, the strips were incubated with 1:2,000 rabbit IgG-HRP conjugate (Jackson ImmunoResearch Laboratories, Inc.) in binding buffer for 1 h at 25°C. The presence of bound HRP was detected by chemiluminescence.
Coimmunoprecipitation immuoblot experiments
For each strain 0.5 g of cell powder was used. To adjust for total protein amount, 0.5 g of untagged cell powder was added to strains marked "coexpressed." The powder was thawed into 5 ml of EB1 supplemented with 300 mM NaCl, 1 mg/ml Heparin for Spc42pPrA, and 150 mM NaCl, 1 mg/ml Heparin for Spc110pPrA, cleared by centrifugation and incubated with 7.5 mg of rabbit IgGconjugated Dynabeads. In all cases the lysates were rotated with the beads for 2 h at 4°C and subsequently treated as described for affinity PrA purification. Samples of each cell lysate and each isolated complex were resolved in duplicate by SDS PAGE and transferred onto nitrocellulose. The presence of PrA and Myc in the samples was detected by immunoblotting using a 1:1,000 dilution of a rabbit IgG (ICN Biomedicals) and a mouse monoclonal antiMyc antibody (Santa Cruz Biotechnology, Inc.).
Microscopy
For in vivo fluorescence experiments cells expressing DsRed-, GFP-, CFP-, or YFP-tagged proteins were grown overnight in YPD or the appropriate selective medium containing 200 µg/ml adenine to reduce autofluorescence. The next morning cells were diluted in the same medium and grown to mid-log phase for 45 h. When ready, cells were harvested and concentrated in SC medium with 200 µg/ml adenine. A small drop (12 µl) of the concentrated cell suspension was spotted on a poly-L-lysinecoated slides before immobilization using a coverslip. Cells were observed immediately after immobilization at 25°C. Cell were visualized with a 100x 1.4 numerical aperture Planapochromat objective using an inverted Carl Zeiss Axiovert 200 wide-field confocal microscope fitted with a Perkin-Elmer Ultra-View spinning disk confocal head on side-port optimized for real-time confocal imaging. The microscope was equipped with a Hamamatsu Orca ER-cooled CCD camera. Image analysis was performed using the MetaMorph software provided by Universal Imaging Corp. For three-dimensional volume and surface reconstruction we used the Imaris and Imaris-Surpass (Bitplane AG) software.
For DAPI staining, cells were grown to mid-log phase in YPD containing adenine as above. Cells were fixed in 4% parafomaldehyde, 3.4% sucrose, and 0.1 M KPO4 for 515 min at 25°C. Cells were washed with 1.2 M sorbitol, 0.1 M KPO4, pH 7.5, sonicated, and subsequently permeabilized with 0.1% Triton X-100 in the same buffer for 1 min at 25°C. Cells were stained with 0.06 µg/ml DAPI in sorbitol/KPO4 for 10 min at 25°C, washed in the same buffer, and immobilized on slides. For image acquisition we used an Axioplan 2 microscope (Carl Zeiss MicroImaging Corp.), with a 100x 1.4 numerical aperture Planapochromat objective, fitted with a Hamamatsu C4742-95 cooled charge-coupled device camera (Sciscope Instrument) interfaced with the OpenLab software (Improvision).
Samples were prepared for thin-section TEM essentially as described (Rout and Blobel, 1993). For postembedding labeling immuno-EM samples were prepared essentially as reported (Wente et al., 1992). The primary antibody was a 1:2,000 dilution of a polyclonal rabbit anti-GFP raised against the whole GFP molecule. The secondary antibody was 10 nm gold particles conjugated goat antirabbit IgG.
Quantitative image analyses
All the quantitative analyses were performed on digital images. Scoring of SPB numbers on DAPI-stained nuclei was performed on two-dimensional projections of three-dimensional image stacks containing 8 0.35 µm optical sections after overlaying them onto differential interference contrast images for clearer positioning of SPB signals and nuclei. Scoring of the colocalization between Spc42pCFP signal versus YFPMlp1p was performed on two-dimensional maximum projections of three-dimensional image stacks containing 1015 0.27 µm optical sections. Only nuclei that presented with a typical C-shaped Mlp distribution (i.e., nuclei in which the nucleolus was positioned around the mid-plane of the stack) were scored for the localization of the Spc42pCFP signal. All other analyses were performed directly using Z-calibrated three-dimensional digital image stacks, which contained 1520 0.27 µm optical sections. Scoring of spindle morphology frequencies in live cells expressing GFPTub1p was performed using the Manually Count Objects module included in the MetaMorph software. All length measurements were obtained using the Measure XYZ Distance module of the same software. The spindle migration index of a cell is proportional to the proximity of its spindle to the bud neck and is calculated as the minimal distance between the neck and the nearest edge of spindle, divided by the maximal mother cell axis (i.e., distance between the bud neck and the most distal edge of the mother cell; Jacobs et al., 1988).
Differential elutriation
Haploid strains yMN291 and yMN293 and diploid strains yCS101 and yCS251 were subjected to differential elutriation as previously described (Miller and Cross, 2001). 12 fractions were collected from each experiment. Cells were harvested by filtration and prepared for either DAPI staining and fluorescence microscopy (yMN291 and yMN293), for thin section EM analysis (yCS101 and yCS251) or for postembedding-labeling EM (yMN291 and yMN293). Progression of cells through the cell cycle was monitored by budding index and by FACS (Becton Dickinson) as described previously (Epstein and Cross, 1992).
Online supplemental material
Fig. S1 shows the distribution of YFPMlp2p in comparison to the nucleolus and the NPCs by fluorescence microscopy. Fig. S2 shows the budding state and the DNA content of the elutriation fractions used in Fig. 5. Fig. S3 shows the repression of PrA-tagged Mlp2p in dextrose containing media to levels below the detection limit by immunoblotting. Fig. S4 shows the growth effects of depleting or deleting the Mlp proteins in a series of SPB temperature sensitive mutant backgrounds. Table S1 shows the S. cerevisiae strains used in this study. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200504140/DC1.
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Acknowledgments |
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This work was supported by a grant from the American Cancer Society (RSG0404251), an Irma T. Hirschl Career Scientist Award, a Sinsheimer Scholar Award, and a grant from the Rita Allen Foundation to M.P. Rout; by grants from the National Institutes of Health to M.P. Rout (GM062427), B.T. Chait (RR00862), and B.T. Chait and M.P. Rout (CA89810); and by a fellowship from the Boehringer Ingelheim Fonds to M. Niepel.
Submitted: 26 April 2005
Accepted: 15 June 2005
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