(A) Diagram of Spc110's functional domain organization and the position of phospho-sites investigated in this study. These sites are located at the N-terminal domain of Spc110, which directly interacts with γ-TuSC (). The conserved centrosomin motif 1 (CM1) (; ) is also within the N-terminal domain. The C-terminal domain of Spc110 is involved in the interaction with SPB central plaque components Spc29 and Spc42 (; ). CBD: calmodulin binding domain. (B) Post-translational modifications of Spc110^1–220 are required for promoting γ-TuSC oligomerization. Spc110^1–220 was expressed and purified from insect cells (middle panel) or from E. coli (right panel). Only Spc110^1–220 from insect cells carried post-translational modifications (). Recombinant γ-TuSC was incubated with Spc110^1–220 or TB150 buffer only on ice. Oligomerization of γ-TuSC-Spc110^1–220 was tested by gel filtration chromatography using a Superdex 200 10/300 column. Peak fractions of the chromatograms were analysed by SDS-PAGE and silver staining.
DOI: http://dx.doi.org/10.7554/eLife.02208.003

(A) Table of mass-spectrometry identified phosphopeptides of Spc110^1–220 purified from baculovirus-insect cell expression system. (B) Mass spectra of identified Mps1 sites (S60 and T68). (C) Mass spectra of identified Cdk1 sites (S36 and S91). (D) Comparison of investigated phospho-sites of Spc110 N-terminal domain with published data (; ; ; ; ).
DOI: http://dx.doi.org/10.7554/eLife.02208.004

(A–C) Purified γ-TuSC (A) spontaneously oligomerizes in BRB80 buffer (B) but not in TB150 buffer (C). Proteins were analyzed by Superose 6 10/300 and subsequently gel filtration fractions were subjected to SDS-PAGE and silver staining. Samples in the peak fraction were subjected to negative staining and protein complexes were analyzed by electron microscopy. Corresponding scale bars are shown on the images.
DOI: http://dx.doi.org/10.7554/eLife.02208.005

(A) Summary of combination of non-phosphorylatable and phospho-mimicking mutations in Spc110 used in this study. The indicated Spc110^1–220 variants were expressed, purified from E. coli, and then tested for γ-TuSC oligomerization. WT: wild-type; 2A/D: S36A/D, S91A/D; 3A/D: S60A/D, T64A/D, T68A/D; 5A/D: S36A/D, S91A/D, S60A/D, T64A/D, T68A/D (see for SDS-PAGE of purified Spc110^1–220 proteins and for gel filtration chromatograms). (B) Spc110^1–220 phospho-mimicking proteins induced γ-TuSC oligomerization. Spc110^1–220 proteins were incubated with γ-TuSC in TB150 buffer. The reconstituted complexes were separated according to size by gel filtration using a Superose 6 column. (C) Bar graph of area ratio of void-volume peak to total area of chromatogram of (B). ** marks statistical significance at p<0.01. Error bars represent SEM. N = 3 to 9 for the number of experiments performed. (D) Void-volume peak fractions of (B) were subjected to negative staining and protein complexes were analyzed with electron microscopy. Representative ring-like structures of γ-TuSC-Spc110^1–220-2D, γ-TuSC-Spc110^1–220-3D, and γ-TuSC-Spc110^1–220-5D. See for additional EM pictures. Scale bar: 50 nm. (E) Quantification of (D). Shown is the particle number per field. Particles were categorized based on the morphology. N is indicated on the figure for the number of fields analysed. (F) Quantification of void-volume fractions of γ-TuSC chromatograms. γ-TuSC was incubated with Spc110^1–220-WT, Spc110^1–220-5D, Spc110^1–220-T18A, Spc110^1–220-T18D, and Spc110^{1–220-5D−T18D} as described in (B). Bar graph of area ratio of void-volume peak to total area of chromatogram was calculated as in (C). ** marks statistical significance at p<0.01. Error bars represent SEM. N = 3 to 9 for the number of experiments performed. (G) Quantification of EM. Void-volume peak fractions of (F) were subjected to negative staining and protein complexes were analyzed with electron microscopy. Particles were categorized based on the morphology. Shown is the particle number per field. Note, the Spc110^1–220−WT and Spc110^1–220-5D graphs are the same as in (E). N is indicated on the figure for the number of fields analysed. (H) Multiple sequence alignment of SPM element of γ-complex receptors from yeast to human. Residues are marked according to the ClustalX colour scheme. The occurrence of each amino acid in each position of CM1 motif is presented with Weblogo 2.0. (I) The indicated Spc110^1–220 proteins (Spc110^1–220-5D, Spc110^{1–220-5D-CM1-QA}, and Spc110^{1–220-5D-ΔSPM}) were incubated with γ-TuSC in TB150 buffer. The reconstituted complexes were separated according to size by gel filtration using a Superose 6 column.
DOI: http://dx.doi.org/10.7554/eLife.02208.006

(A) Purified GST-Spc110^1–220 variants. ∼1 μg of protein was loaded to each lane. The SDS-PAGE was stained with Coomassie Blue. (B) Calibration of Superose 6 10/300 gel filtration column. Following protein markers were used: thyroglobulin (667 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa). Partition coefficient (K_av) was calculated using the equation: (V_e–V₀)/(V_c–V₀), where V₀ = column void volume, V_e = elution volume, and V_c = geometric column volume. The calibration curve (bottom) of K_av vs log molecular weight was plotted in semi-logarithmic scale. (C) Gel filtration chromatograms of purified GST-Spc110^1–220 variants. Proteins were analyzed in TB150 buffer by Superose 6 10/300 chromatography.
DOI: http://dx.doi.org/10.7554/eLife.02208.007

(A) Overlapped gel filtration chromatograms with molecular weight markers. The peak of the void-volume (V₀, boxed area) corresponds to molecular weight fractions higher than 5000 kDa. Note that the concentrations of γ-TuSC and Spc110^1–220 variants in (A) were double as high compared to (B) and (C). (B) Gel filtration chromatograms of γ-TuSC incubated with phosphomimetic or non-phosphorylatable Spc110^1–220 mutant proteins. γ-TuSC was incubated in TB150 buffer with Spc110^1–220 proteins from . Complexes were analyzed by Superose 6 10/300 chromatography. Fractions corresponding to the peak were analyzed by SDS-PAGE and silver staining. (C) As (B) but with Spc110^1–220 variants that have defective SPM due to T18D or T18A.
DOI: http://dx.doi.org/10.7554/eLife.02208.008

Gel filtration chromatograms of γ-TuSC incubated with indicated Spc110^1–220 variants in TB150 buffer. Non-phosphorylatable mutations in addition to phosphomimetic mutations were introduced on Spc110^1–220 to examine the possibility that non-phosphorylatable mutations alter overall structure and thus disrupt the activity to induce γ-TuSC oligomerization. Complexes were analyzed by Superose 6 10/300 chromatography.
DOI: http://dx.doi.org/10.7554/eLife.02208.009

(A) Additional examples of ring-like and filament-like γ-TuSC-Spc110^1–220 complexes. Scale bar: 50 nm. (B) Summary of particle numbers of γ-TuSC oligomerized by Spc110^1–220 phospho-mimicking variants. Both the absolute particle number and the percentage of each particle category are shown. (C) Spc110^1–220-2D, Spc110^1–220-3D, and Spc110^1–220-5D induced similar level of ring-like γ-TuSC-Spc110^1–220 complexes. * marks statistical significance at p<0.05. Error bars represent SEM. N = 15–25 for the number of field analyzed. Correlated to . (D) The diameter of the ring-like γ-TuSC-Spc110^1–220 complexes showed no difference among Spc110^1–220 phosphomimetic variants. Error bars represent SEM. N = 9, 12, and 18 for the number of ring-like complexes measured in 2D, 3D, and 5D, respectively.
DOI: http://dx.doi.org/10.7554/eLife.02208.010

(A) Gel filtration chromatograms of γ-TuSC incubated with phosphomimetic Spc110^1–220-5D with additional T18 mutations. Complexes were analyzed by Superose 6 10/300 chromatography in TB150 buffer. (B) Growth of ten-fold serial dilutions of SPC110 shuffle strains carrying the empty integration vector (null) or SPC110 (WT) or SPC110 mutant alleles on the integration vector. Wild type and SAC deficient mad2Δ cells were analyzed. Growth was tested either on synthetic complete (SC) plates containing 5-FOA or SC dropout plates.
DOI: http://dx.doi.org/10.7554/eLife.02208.011

Multiple sequence alignment of selected γ-TuSC receptor family members containing CM1 motifs. Two of the most conserved residues within CM1 motif are marked with asterisks and mutated to disrupt CM1 function in the spc110^CM1-QA mutant. The occurrence of each amino acid in each position of CM1 motif is presented with Weblogo 2.0.
DOI: http://dx.doi.org/10.7554/eLife.02208.012

(A) GST pull-down assays were performed between γ-TuSC (containing His-tagged Spc97-6His and Spc98-6His) and the GST-tagged Spc110^1–220 proteins. The bound proteins were eluted with sample buffer and separated on SDS-PAGE and analyzed by immunoblotting with anti-His, anti-GST, and anti-Tub4 antibodies. Infrared-dye-labelled secondary antibodies were applied and detected with Li-cor imaging system. (B) Quantification for bound Spc97, Spc98, and Tub4 from (A) at 300 nM Spc110^1–220. ** marks statistical significance at p<0.01. Error bars represent SEM. N = 3 for the number of experiments performed. (C) Mutations in SPM or CM1 affect γ-TuSC binding. As (A) but performed with the indicated SPM and CM1 mutant Spc110^1–220 constructs. (D) Quantification of Spc110^1–220 variants and bound Spc97, Spc98, and Tub4 from (C) at 300 nM Spc110^1–220. ** marks statistical significance at p<0.01 and *** at p<0.001. Error bars represent SEM. N = 3 for the number of experiments performed.
DOI: http://dx.doi.org/10.7554/eLife.02208.013

(A) Enhancement of MT nucleation activity of Spc110 by Mps1 and Cdk1 phosphorylation. Representative image fields of Alexa546-labeled microtubules from the nucleation assay polymerized in the presence of buffer, γ-TuSC and Spc110^1–220 variants (B). Scale bar: 10 µm. (B) Quantification of the MT nucleation assay. For each experiment, the number of MTs was counted from 20 fields. The MTs/field was normalized with MTs/field obtained by the MT nucleation reaction in the presence of buffer only. N = 6 to 9 for the number of independent experiments performed. * marks statistical significance at p<0.05 and ** at p<0.01. Error bars represent SEM.
DOI: http://dx.doi.org/10.7554/eLife.02208.014

(A and B) Two phospho-specific antibodies were generated from guinea pigs to recognize phosphorylation of Cdk1 sites (pS36-pS91) (A) and Mps1 sites (p60-p64-pT68) (B). In vitro kinase assays were performed in the presence of recombinant Mps1 or Cdk1^as1 and Spc110^1–220, either wild type (WT) or non-phosphorylatable variants as indicated. 3A (Cdk1) indicates T18A-S36A-S91A and 3A (Mps1) S60A-T64A-T68A. In vitro phosphorylated Spc110^1–220 was subjected to SDS-PAGE and immunoblot with the corresponding phospho-specific antibodies. The specific kinase activity of Cdk1^as1-Clb2 and Cdk1^as1-Clb5 was compared using human histone H1 as substrate (A, bottom). The numbers in the histone H1 experiment represent the relative kinase activity. As negative control of kinase activity, Mps1 was inactivated with chemical inhibitor SP600125 (B), while Cdk1^as1-Clb2 and Cdk1^as1-Clb5 overexpressed and purified from budding yeast were inactivated with 1NM-PP1 (A). (C) Phosphorylation of Spc110 and Spc110^5A in vivo. SPC110-GFP wild type cells (WT) and spc110^5A-GFP cells were arrested in mitosis with nocodazole. Spc110-GFP was enriched using GFP binder conjugated to beads, and the bound proteins were subject to immunoblotting with the indicated antibodies. (D) SPC110-GFP wild type cells were arrested in G1, S phase, and mitosis as indicated. Spc110-GFP was enriched with GFP-binder and analyzed by immunoblotting with the indicated P-specific antibodies. A non-specific band was used as loading control.
DOI: http://dx.doi.org/10.7554/eLife.02208.015

(A) Phospho-specific antibody against phospho-T18 (pT18) was generated from guinea pigs. In vitro kinase assays were performed in the presence of Cdk1^as1 and Spc110^1–220, either wild type (WT) or non-phosphorylatable Spc110^{1–220-3A (Cdk1)} (T18A-S36A-S91A). In vitro phosphorylated Spc110^1–220 was subjected to SDS-PAGE and immunoblot with the phospho-specific antibody. As negative control of kinase activity, Cdk1^as1-Clb2 and Cdk1^as1-Clb5 overexpressed and purified from budding yeast were inactivated with 1NM-PP1. Note that the same Spc110^1–220 samples were analyzed in with the anti-pS36-pS91 antibody. The Spc110^1–220 blot on the bottom is the same as in . (B) Quantification of tryptic phospho-T18 peptide (NMEFpTPVGFIK). Data were acquired in a data independent fashion in a pseudo MRM experiment in the Orbitrap mass spectrometer fragmenting peptides with m/z = 689.81. Extracted ion chromatogram of phosphorylated NMEFTPVGFIK was quantified. Expected retention time of the tryptic phospho-T18 peptide is 24.7 min. (C) Mass spectrum of fragmented phospho-T18 peptide. The graph summarizes the identified fragmented ions. (D) Gel filtration chromatograms of γ-TuSC. Spc110^1–220-5D was incubated with Cdk1^as1-Clb2 in the presence (+1NM-PP1) and absence of the Cdk1^as1 inhibitor 1NM-PP1 and ATP. This allowed Spc110^T18 phosphorylation in the absence but not in the presence of 1NM-PP1. Adding 1NM-PP1 stopped all Cdk1^as1 kinase reactions. The so modified Spc110^{1–220-5D-pT18} was then incubated with γ-TuSC. γ-TuSC was analysed by gel filtration using a Superose 6 column for oligomerization. The gel filtration fractions were analyzed by immunoblotting for Spc97/Spc98, Tub4, Spc110^1–220, and Spc110^{1–220−pT18} distribution. The anti-pT18 blot on the bottom is a 25-times of contrast enhancement of the blot on top. Fractions covering the void-volume peak were marked with a red rectangle outline. (E) Quantification of void-volume fractions shown in (D). Spc110^{1–220-5D-T18D} was included as control. Indicated protein band intensities of fractions covering the void-volume peak (red rectangle outline in (D)) were summed and divided by the total intensity of fractions. * marks statistical significance at p<0.05, ** at p<0.01, and *** at p<0.001. Error bars represent SEM. N = 4 for the number of experiments performed.
DOI: http://dx.doi.org/10.7554/eLife.02208.016

(A) Growth of 10-fold serial dilutions of SPC110 shuffle strains with integration vector encoding SPC110 (WT) or SPC110 mutants with and without the SAC gene MAD2. Growth was tested either on synthetic complete (SC) plates containing 5-FOA or SC dropout plates. (B) The indicated SPC110 wild type cells and spc110 mutant cells carrying GFP-TUB1 SPC42-mCherry were incubated at 37°C, the restrictive temperature of some of the spc110 mutants (). The fluorescent signal of GFP-Tub1 at SPBs was quantified in G1 phase cells (no buds), early S phase cells (small bud with unsplit SPBs) and M phase cells (large bud with split SPBs and spindle length within 2 μm). 50 cells were analyzed per cell cycle phase and strain. Error bars represent SEM. * marks statistical significance at p<0.05, ** at p<0.01, and *** at p<0.001. N = 2 or 3 for the number of experiments performed. (C) The representative metaphase cells from (B) were scanned for the distribution of the GFP-Tub1 and the Spc42-mCherry signal along the spindle axis. Scale bar: 4.5 µm. (D) In vivo MT re-nucleation assay. Wild-type SPC110 cells (WT) and the indicated spc110 cells with GFP-TUB1 were treated as indicated in the outline. The GFP-Tub1 signal at SPBs was measured at 0, 30, and 60 min after nocodazole washout. N = 40 for the number of cells analyzed per time point and strain. Error bars represent SEM. (E) Data of (D) after 30 and 60 min. Error bars represent SEM. *** marks statistical significance at p<0.001.
DOI: http://dx.doi.org/10.7554/eLife.02208.017

(A) There was no significant difference in the protein levels of Spc110 variants. Asynchronous cells grown to OD₆₀₀ 0.5 were harvested and subjected to TCA extraction for whole cell protein lysates. SDS-PAGE and immunoblotting with anti-Spc110 were performed. Tub2 was loading control. (B) The representative images of wild type SPC110 (WT) and spc110 cells in G1 (no bud) and S phase (small bud). Microtubules were marked with GFP-Tub1 and SPBs were marked with Spc42-mCherry. See for experimental set-up. BF: bright field. Scale bar: 4.5 µm. (C) The indicated wild type SPC110 (WT) and spc110 cells were grown at 37°C. The cell cycle stages were determined according to cell morphology and spindle length. Unfixed cells were analyzed for Spc97-GFP signal at SPBs. N = 30 for the number of cells analyzed for each category. * marks statistical significance at p<0.05 and ** at p<0.01. Error bars represent SEM. (D) SPC110 and spc110^T18D cells with SPC97-GFP were analyzed by time lapse analysis. The fluorescent intensity was recorded and measured over time (top). Error bars represent SEM. N = 17 for the number of cells analysed for each strains. The Spc97-GFP signals of selected cells are shown on the bottom. Scale bar: 5 μm. (E) The indicated wild type SPC110 (WT) and spc110 cells were grown at 37°C. The cell cycle stages were determined according to cell morphology and spindle length. Unfixed cells were analyzed for Spc110-GFP signal at SPBs. Error bars represent SEM. N = 30 for the number of cells analyzed for each category. (F) Similar experiment as performed in . The indicated SPC110 wild type and spc110^ΔSPM carrying GFP-TUB1 SPC42-mCherry were incubated at 37°C. The fluorescent signal of GFP-Tub1 at SPBs was quantified in G1 phase cells (no buds), early S phase cells (small bud with unsplit SPBs) and M phase cells (large bud with split SPBs and spindle length within 2 μm). 100 cells were analyzed per cell cycle phase and strain. Error bars represent SEM. ** marks statistical significance at p<0.01, and *** at p<0.001. (G) Similar experiment as performed in (D) with SPC110 and spc110^ΔSPM cells. SPC110 and spc110^ΔSPM cells with SPC97-GFP were analyzed by time lapse analysis. Error bars represent SEM. N = 17 for the number of cells analysed for each strain. (H) Similar experiment as performed in (E). The wild type SPC110 (WT) and spc110^ΔSPM cells were grown at 37°C. The cell cycle stages were determined according to cell morphology and spindle length. Unfixed cells were analyzed for Spc110-GFP signal at SPBs. Error bars represent SEM. N = 40 for the number of cells analyzed for each category.
DOI: http://dx.doi.org/10.7554/eLife.02208.018

(A) Alignment of the amino acid sequence of GCP3 homologues from yeast to human. Shown are the putative α-helical regions H1–H5. Residues are marked according to the ClustalX colour scheme. (B) Growth test of spc98^Δ1–156 cells with and without the SAC gene MAD2 at 23°C and 37°C. “SPC98” indicates the spc98ΔSPC98 cells while YPH499 is the unmodified wild type strain. (C) GFP-Tub1 signal at SPBs in SPC98 wild type or spc98^Δ1–156 cells. The experiment was performed as in . ** marks statistical significance at p<0.01. N = 100 cells analysed for each strain. (D) SPC98 and spc98^Δ1–156 cells with SPC42-mCherry, SPC97-GFP, or SPC110-GFP were analyzed by time lapse analysis. The fluorescent intensity was measured over time (right). t=0 for the time point of SPB separation. Error bars represent SEM. N = 14 and 19 for the number of cells analysed in SPC97-GFP and SPC-110-GFP strains respectively. The Spc97-GFP and Spc110-GFP signals of selected cells are shown on the left panel. Scale bar: 5 μm. (E) Spc110^5D combined with γ-TuSC^{Spc98Δ1–156} fails to induce oligomerization in TB150 buffer. The experiment was performed as in . The purified γ-TuSC^{Spc98Δ1–156} after SDS-PAGE and Coomassie Blue staining is shown with relative intensity of the protein bands. The protein distribution in the Superdex 200 10/300 chromatogram was analyzed with SDS-PAGE and silver staining. (F) Binding of γ-TuSC and γ-TuSC^{Spc98Δ1–156} to Spc110^1–220 mutant proteins. The binding reaction was performed and analyzed by immunoblotting as described in . (G) Quantification of . Pull-downed Spc97, Spc98, and Tub4 proteins at 300 nM Spc110^1–220-5D. Error bars represent SEM. N = 3 for the number of experiments performed. *** marks statistical significance at p<0.001.
DOI: http://dx.doi.org/10.7554/eLife.02208.019

(A) Structure and functional domain organization of budding yeast Spc98 and human GCP3. Both Spc98 and GCP3 share the conserved N-terminal domain (NTD), the GCP core body and the divergent, unstructured linker connecting NTD and GCP core body. The GRIP1 region is within the region interacting with Spc97, while the GRIP2 covers the region interacting with Tub4 (; ; ; ). (B) The growth test of MAD2 or mad2Δ cells with spc98 N-terminal truncated mutant alleles. Serial diluted cells were spotted on YPAD plates and incubated at indicated temperatures for two or three days. SPC98 indicates the spc98Δ SPC98 cells while WT is the unmodified wild type strain. The design of each truncated variant is shown in the cartoon below. (C) The representative images of spc98^Δ1–156 cells in M phase (large budded, bipolar spindle within 2 μm long). SPC98 wild type and spc98^Δ1–156 cells with SPC42-mCherry GFP-TUB1 were grown at 37°C for 2 hr. Metaphase-like cells were analyzed for the presence of bipolar spindles. BF: bright field. Scale bar: 4 μm. (D) Quantification of MT phenotype of metaphase cells of (C). One hundred SPC98 and spc98^Δ1–156 cells with a large bud and a SPB distance of ∼2 µm were categorized into ‘normal spindle’ or ‘immature/defective spindle’.
DOI: http://dx.doi.org/10.7554/eLife.02208.020

(A) γ-TuSC^{Spc98Δ1–177} was purified from insect cells, incubated in TB150 buffer, and then analyzed by Superose 6 10/300. The left panel presents the Coomassie Blue stained SDS-gel of purified γ-TuSC^{Spc98Δ1–177}. (B) γ-TuSC^{Spc98Δ1–177} in fraction 7 from (A) were subjected to negative staining and analyzed with electron microscopy. The particles showed irregular morphology, suggesting the formation of protein aggregates. Corresponding scale bars are shown in the images. (C) Similar experiment as performed in (A). Purified γ-TuSC^{Spc98Δ1–156} was incubated in TB150 or BRB80 buffer and then analyzed by Superose 6 10/300. γ-TuSC^{Spc98Δ1–156} oligomerized in BRB80 but not in TB150 buffer. (D and E) Similar experiment as performed in (B). After the gel filtration shown in (C), the peak fraction of γ-TuSC^{Spc98Δ1–156} (fraction 7 in BRB80 and fraction 13 in TB150) was subjected to negative staining and analyzed with electron microscopy. Corresponding scale bars are shown on the images.
DOI: http://dx.doi.org/10.7554/eLife.02208.021

(A) Graphical representations of the patterns of CM1 motif within the multiple sequence alignment of γ-TuCR protein sequences. The CM1 motif sequence logos were shown for γ-TuCR protein sequences retrieved from Pfam database (Microtub_assoc, Pfam id: PF07989), Spc110s and Spc72s from subphylum Saccharomycotina. The Spc72 sequences used to generate the short CM1 logo are shown in . The sequence logos were generated with Weblogo 3.0 server. The conserved residues shared by CM1 defined by Pfam, Spc110s, and Spc72s are marked with asterisks. (B) SPM and CM1 in PCNT, Spc110, and Pcp1 homologues (SPM-CM1 type) and in CDK5RAP2/Cnn/Spc72/Mto1 (CM1 type). The occurrences of the motif in the training set sequences were calculated and shown as coloured blocks on a line with MEME motif scanning analysis (). Weblogo motif diagram is shown for each sequence showing the sites contributing to that motif in that sequence. The best p-value for the sequence/motif combination is listed. The p-value of an occurrence is the probability of a single random subsequence with the length of the motif, generated according to the zero-order background model, having a score at least as high as the score of the occurrence.
DOI: http://dx.doi.org/10.7554/eLife.02208.022