Mlp2p interact with the core of assembled SPBs. (a) Mlp2p, but not Mlp1p, strongly interacts with SPB proteins. PrA-containing complexes were affinity purified from cell lysates expressing Mlp1p–PrA, Mlp2p–PrA, or PrA alone as a control. The complexes were separated by SDS-PAGE, visualized by Coomassie staining, and identified by tandem mass spectrometry. Open circles indicate the position of rabbit IgG heavy chain from the Dynabeads. (b) Mlp2p directly binds Spc110p, Spc42p, and Spc29p, but not Cnm67p. Mlp2p–PrA-associated SPB components were affinity purified as described above and eluted from IgG-Dynabeads using 1 M MgCl₂ leaving Mlp2p–PrA attached to the beads. Proteins in the eluate were then resolved by SDS-PAGE and transferred to nitrocellulose. The blot was stained with amido black, cut into strips, and probed with either purified Mlp1p–PrA or Mlp2p–PrA, or with blocking buffer alone. Bound PrA probes were detected with rabbit IgG–HRP conjugate followed by chemiluminescence. The identity of the minor bands has not been determined. (c to f) Mlp2p interacts with assembled SPB cores. Dynabeads carrying Mlp1p–PrA (c) or Mlp2p–PrA (d–f) containing complexes were prepared exactly as described above from either haploid (c–e) or diploid (f) cells and visualized by TEM. Small arrows, haploid SPBs. The electron-dense globular particles visible in panels c and d are also present in empty control beads and are the magnetic material within the Dynabeads. (e) Three representative examples of haploid SPBs bound to Mlp2p–PrA beads at higher magnification. (f) Three examples of diploid SPBs bound to Mlp2p–PrA beads. The diameter and thickness of the central plaque of both haploid and diploid Mlp2p–PrA bound SPBs were measured on calibrated digital images using the ImageJ software and are indicated as the mean value ± 1 SD at the bottom right. Also indicated (**) is the t test P value obtained for the comparison of the distribution of diameters of haploid versus diploid SPBs. Large arrows, filaments attaching the SPBs to the beads. Bars: (c and d) 500 nm; (e and f) 100 nm.

SPB components coimmunoprecipitate Mlp2p, but not Mlp1p. PrA containing affinity-purified complexes (IP) and whole cell lysates (lysate) of strains coexpressing combinations of PrA-tagged SPB components as baits and Myc-tagged Mlp1p or Mlp2p as targets, were probed by immunoblotting for Myc and PrA (coexpressed). To control for interactions occurring after lysis, strains expressing either one PrA-tagged bait or one Myc-tagged target were mixed after cell lysis and analyzed exactly as above (mixed).

The Mlps occupy the same NE hemisphere as the SPB at all stages of the cell cycle. (a) Mlp1p and Mlp2p have different localization patterns. Typical images of homozygous diploid cells expressing both CFP–Mlp1p (red) and YFP–Mlp2p (green). (b) Live fluorescence images of haploid cells expressing either Mlp1p–YFP or YFP–Mlp2p (green) and Spc42-CFP (red). (c) YFP–Mlp2p (green) and Spc42p–CFP (red) double-tagged haploid cells were subjected to differential elutriation to enrich for cells at different stages of the cell cycle. Representative fluorescence images for each cell cycle stage are displayed as indicated. (Merge) Two-dimensional image projections for both YFP and CFP were overlaid over a DIC images of each individual cell. (3D) A three-dimensional rendition of each double-color image was obtained using the Imaris software and overlaid over a digital outline of the whole cell. (a and b) Images represent two-dimensional maximum projections of z-stacks containing 10–15 0.3 μm sections. To visualize the fluorescent signals in the context of the whole cell, a DIC image was merged with both of the CFP and YFP pictures from the same field (Merge). Bars: (a and c) 2 μm; (b) 1 μm.

mlp2Δ cells display a significant delay in proceeding past metaphase and accumulate with short spindles at the bud neck. All experiments were done on asynchronous cell cultures. (a) Images of haploid wild-type, mlp1Δ, and mlp2Δ cells expressing GFP–Tub1p. Images were obtained as described for Fig. 3. Asynchronous cells (n = 300–400) were scored for short and stubby spindles (i.e., cells in S or early M phase) in two independent experiments, to assess their ability to progress through the cell cycle. The numbers of 38%, 39%, and 52% have SDs of 0.3%, 3.0%, and 4.0%, respectively. (b) Tukey plots displaying the spindle migration index distributions for wild-type and mutant cells lacking either Mlp1p or Mlp2p. Only cells with a metaphase spindle appearance were used for this analysis (n > 60). The top and bottom of each rectangle represent the 75th and 25th percentiles, respectively; the center bar of the rectangle marks the median spindle migration index value. The top and bottom horizontal marks show the 90th and 10th percentiles, respectively. Black dots represent outlying data points. The distributions were not found to be significantly different based on individual pairwise comparisons using the t test. (c) Images of haploid wild-type, mlp1Δ, and mlp2Δ cells expressing Spc42p–GFP. (d) Tukey plots displaying the SPB-to-SPB distance distributions in wild-type and mutant cells lacking either Mlp1p or Mlp2p. Only cells between S and early M were used for this analysis. The median SPB-to-SPB distance was 1.58 μm (n = 60) in wild-type cells, 1.35 μm (n = 54) in mlp1Δ cells and 1.10 μm (n = 120) in mlp2Δ cells. Comparisons of the distributions using the t test showed a high degree of significance in all cases: wild type versus mlp1Δ, P < 0.0003; wild type versus mlp2Δ, P < 0.0001; mlp1Δ versus mlp2Δ, P < 0.0025. Bar, 2 μm.

mlp2Δ cells accumulate multiple amorphous nuclear Spc42p-containing bodies that nucleate microtubules. (a) Quantitative analysis of the number of Spc42p–GFP containing foci in wild-type, mlp1Δ, and mlp2Δ cells. Asynchronous Spc42p–GFP haploid cells were fixed and stained with DAPI to reveal the position of the nucleus. Two-dimensional image projections of three-dimensional image stacks obtained for both GFP and DAPI were overlaid onto each respective DIC image for scoring. We counted the number of cells with multiple Spc42p–GFP signals (n > 600). (b) Wild-type, mlp1Δ, and mlp2Δ cells expressing Spc42–GFP were separated on the basis of cell sizes by differential elutriation (see Fig. S2). For each sample, cells present in equivalent elutriation fractions were imaged as described for panel a to reveal the presence of Spc42p–GFP (red) and of the DAPI-stained nucleus (blue). Two representative fields of vision are presented for mlp2Δ. Bar, 2 μm. (c) Left panels, thin section, TEM images of diploid mutant cells lacking Mlp2p, after enrichment for large-budded cells and cell chains by elutriation. Arrowheads, amorphous electron-dense material presumably composed of misassembled SPB components. Arrows, spurious microtubule bundles emanating from MTOC positioned perpendicular to the visible SPB. (Inset) Twofold magnification of an area presented in the bottom left panel, showing one of these bundles of microtubules. Right panels, line drawings of the panels shown on the left highlighting the position of microtubule bundles, the NE, SPBs, and amorphous electron-dense SPB-like structures. (d) Postembedding labeling immuno-EM images of diploid mutant cells expressing Spc42p–GFP. Thin sections from embedded samples were labeled with a rabbit anti-GFP polyclonal antibody followed with 10 nm gold-conjugated secondary antibody. Arrowhead, gold particle–decorated fully formed SPB. Arrows, amorphous intranuclear Spc42p–GFP-containing bodies. The bottom right panel has a twofold magnification with respect to the other panels. Bars, 100 nm.

Low expression levels of MLP2 in conjunction with spc110-220 cause lethality and loss of nuclear integrity at 37°C. (a) Two individual clones of strains expressing either SPC110 or the spc110-220 allele alone (−) or in combination with either MLP1 or MLP2 under the control of the GAL1-10 promoter (Gal-MLP1 and Gal-MLP2, respectively) were spotted in 10-fold dilution steps on dextrose-containing plates and grown for 2 d at the indicated temperatures. (b) DAPI-stained cells of the indicated genotypes were scored for grossly aberrant nuclear morphology (n > 300). (Inset) Typical appearance of cell with altered nucleus. (c) Semiquantitative relative measure of dilution-adjusted colony density from strains with the indicated genotype and their counterparts carrying the wild-type SPB protein allele grown as described in panel a. This analysis was performed as previously described (Tackett et al., 2005). Data presented in panel a and in Fig. S4 was used for this analysis.

Cells lacking Mlp2p have a defect in incorporating newly made components into their SPBs with respect to wild type, resulting in significantly smaller SPBs. (a) Haploid wild-type and mutant cells expressing genomically tagged Spc42p–GFP were analyzed by fluorescence microscopy as in Fig. 4. Two-dimensional maximum projections of three-dimensional image stacks were used for quantitative analysis of the GFP signal associated with each individual SPB. In each experiment we scored 100–200 individual SPBs. We repeated the experiment three times with similar results. Data from each experiment were normalized to the average wild-type intensity value and a combination of all data is presented. The 95% confidence interval for the mean of the distributions of relative SPB intensity measurements for each strain is displayed in the graph. The mean SPB intensities ± SD were: wild type 1.00 ± 0.128 and mlp2Δ 0.915 ± 0.114. The significance of the difference between the two normal distributions was evaluated using the t test and the P value is indicated at the bottom of the graph. (b) Deletion of MLP2 does not influence the expression of Spc42p–GFP. Total cell lysates from haploid mutant or wild-type strains used for the experiment in panel a, were run on SDS-PAGE transferred to nitrocellulose. Blots were probed with anti-GFP monoclonal antibody to determine the levels of expression of Spc42p–GFP and with anti-Pgk1p to control for loading levels. The intensity of the Spc42p–GFP signal in each of the indicated strains were quantified using the OpenLab software and normalized against the Pgk1p signals to produce the graph presented (top). The results indicate no significant difference between mutant and wild-type strains.

Mlp2p links the SPB into the nuclear peripheral Mlp layer and aids the incorporation of new components into its tightly packed core.