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First published online 9 January 2007
doi: 10.1242/jcs.03342

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Phosphorylation of Spc110p by Cdc28p-Clb5p kinase contributes to correct spindle morphogenesis in S. cerevisiae

Stephen M. Huisman, Monique F. M. A. Smeets^* and Marisa Segal

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK

View larger version (59K): [in this window] [in a new window]   Fig. 1. Localisation of Clb5p-GFP in live cells. (A-C) Selected frames from representative time-lapse sequences illustrating Clb5p localisation in wild-type cells. (A) Wild-type cells expressing Clb5p-GFP exhibited diffuse nuclear labelling following bud emergence. In the cell on the right, a prominent dot presumably marking side-by-side SPBs (0 minutes, arrow) gave way to a bar bisecting the nucleus (2-8.5 minutes, arrowheads), consistent with labelling of the mitotic spindle. The cell on the left shows localisation to the nucleus and mitotic spindle (arrowheads indicate the position of the spindle poles). As the spindle inserted at the bud neck, labelling decreased progressively (from 27.5 minutes) before spindle elongation in anaphase began. (B,C) Wild-type cells coexpressing Clb5p-GFP (overlaid in green) and Spc29p-CFP (in red). (B) Initial accumulation of Clb5p-GFP in the nucleus and the SPB (0-10 minutes, arrow) in early S phase. As the SPBs separated, label began to accumulate at the mitotic spindle (31 minutes, arrowheads). (C) Following accumulation at the mitotic spindle (arrowheads), the Clb5p-GFP label progressively disappeared before spindle elongation (right cell, 11 minutes). Numbers indicate time elapsed in minutes. Arrows indicate labelling of the SPB and arrowheads indicate the poles of the mitotic spindle. (D) Representative fluorescence images for Clb5p localisation in cnm67 cells coexpressing Clb5p-GFP (overlaid in green) and Spc29p-CFP (in red). Bars, 2 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Localisation of Spc11013p in wild-type cells. (A) Diagram summarising the key domain features of the SPB component Spc110p (Kilmartin et al., 1993; Geiser et al., 1993; Friedman et al., 1996; Stirling et al., 1996; Adams and Kilmartin, 1999; Elliot et al., 1999; Friedman et al., 2001) and depicting the predicted product of the high-dosage suppressor spc11013. CBD, calmodulin-binding domain; nls, nuclear localisation signal. (B) Localisation of full-length Spc110p-GFP or truncated Spc11013p-GFP in wild-type cells. Pairs of DIC and fluorescence images are shown. Spc110p-GFP localised to the SPBs throughout the cell cycle whereas Spc11013p-GFP localised uniformly to the nucleus. Bars, 2 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Phosphorylation of Spc110p dependent on CDK consensus sites. (A) Western blot analysis of yeast extracts from wild-type cells expressing HA3-Spc110p sampled during a time-course synchrony experiment. Low-mobility bands corresponding to phosphorylated Spc110p appeared as cells initiated bud emergence (45 minutes after the release from G1). Phosphorylation peaked at metaphase (see Friedman et al., 1996). Numbers indicate time in minutes after release from an -factor-induced block; a, extract from asynchronous cells. (B) Effect of overexpression of the phosphatase Cdc14p on Spc110p phosphorylation. An overnight cell culture in YEPRaffinose (R) was synchronised by addition of 15 µg/ml nocodazole (R + Noc). During the arrest, the culture was divided and dextrose (D) or galactose (G) was added for repression or induction of GAL1:CDC14, respectively. Extracts were obtained from cell samples collected after 1, 2 and 3 hours. 90% of cells were held prior to anaphase in all samples analysed (not shown). Asterisk indicates a crossreacting band. (C) Effect of overexpression of Clb5p on Spc110p phosphorylation. Cells of the indicated strains were synchronised in YEPRaffinose by addition of 15 µg/ml nocodazole (R + Noc). Cells were released from the arrest by transferring to either YEPDextrose (D) or YEPGalactose (G) medium to repress or induce GAL1:CLB5, respectively. Extracts were prepared after incubation for 2 or 4 hours. (D) Western blot analysis of extracts from asynchronous cells expressing wild-type Spc110p (WT), Spc11036Ap (S36A), Spc11091Ap (S91A) and Spc11036A91Ap (S36A S91A). c, untagged control. (E) Western blot analysis of extracts from synchronous cell populations, expressing the indicated versions of Spc110p, obtained by release from an -factor-induced arrest. Phosphorylation was delayed in the case of the Spc11091Ap until completion of spindle assembly (75-90 minutes, not shown). Phosphorylation of Spc11036Ap was diminished at later time points reaching a lower maximal level by 75 minutes. The double substituted mutant combined both effects. Cell-cycle analysis accompanying this experiment is shown in supplementary material Fig. S2. HA3-epitope tagged Spc110p and HIS6-tagged Cdc14p were detected as described in Materials and Methods.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Contribution of Mps1p to cell-cycle-dependent phosphorylation of Spc110p. (A) Cell-cycle-dependent phosphorylation of Spc110p following inactivation of Mps1p. The indicated strains were synchronised by -factor-induced block at 23°C and released from G1 at 34°C. The residual cell-cycle-dependent pattern of phosphorylation was dependent on the two CDK consensus sites (and independent of Mps1p). Numbers indicate time in minutes following release from G1. Cell-cycle progression until metaphase was comparable for all strains analysed (not shown). (B) Western blot analysis of extracts from the same synchrony experiment shown in A corresponding to all the strains analysed (and including MPS1+ controls not shown in A) at the 50-minute time point, rearranged for comparison. (C) Cell-cycle-dependent profile of phosphorylation in Spc110p mutants lacking Mps1p phosphorylation sites (mps-) in combination with S36A or S91A substitutions. Extracts prepared from the indicated strains following release from a G1 block. Numbers indicate time in minutes following release from the -factor-induced arrest. Again, phosphorylation was dependent on the two CDK sites in the absence of the previously characterised Mps1p phosphorylation sites (Friedman et al., 2001). a, extract from asynchronous cells. For analysis of cell-cycle progression accompanying this experiment see supplementary material Fig. S3.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Spindle-related phenotypes resulting from substitutions cancelling S36 and S91 putative CDK sites. (A) Distribution of MT-based structures in cell populations of the indicated strains expressing a GFP-Tub1p fusion. At least 500 cells were counted in duplicate experiments. Error bars indicate s.e.m. (B) Cell distribution by spindle length at onset of anaphase B. At least 200 digital images of synchronised cells of the indicated strains expressing GFP-Tub1p were used for measuring spindle length (see Materials and Methods). In wild-type cells the majority of spindles are either shorter than 2 µm or already longer than 3.5 µm as they initiated the fast phase of spindle elongation. Accumulation of spindles in the range of 2-3 µm, in particular, in the spc11091A mutant indicated a slow point or failure to engage in the fast phase of spindle elongation.

View larger version (101K): [in this window] [in a new window]   Fig. 6. Spindle dynamics in wild-type or spc11091A mutant cells at onset of spindle elongation. (A,B) Selected frames from time-lapse series for representative spindle dynamics in wild-type cells expressing GFP-Tub1p. The fast phase of spindle elongation started in a single transition (6-8.5 minutes in A; 2-6 minutes in B). (C,D) Selected frames from time-lapse series for representative spindle dynamics in spc11091A mutant cells expressing GFP-Tub1p. Spindle length increased slowly (4.5-11 minutes in C; 0-6.5 minutes in D) before spindle elongation proceeded at a rate characteristic of the fast phase of wild-type cells. (E) Plot of kinetics of spindle elongation in a wild-type cell vs spc11091A mutant cell. Spindle measurement in digital images from live cell recordings was carried out as previously described (Segal et al., 2000b). Bars, 2 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Effect of a mad2 mutation on spindle dynamics of spc11091A mutants. (A) Cell distribution by spindle length at onset of anaphase B in the indicated strains expressing GFP-Tub1p. Deletion of MAD2 abolished the accumulation of cells exhibiting spindle lengths in the range of 2-3.2 µm. (B) Distribution of MT-based structures in cell populations of the indicated strains expressing a GFP-Tub1p fusion. A MAD2 deletion eliminated the metaphase delay of spc11091A cells. Error bars indicate s.e.m. (C) Plot of kinetics of spindle elongation in spc11091A vs spc11091A mad2. The mad2 mutation allowed an spc11091A cell to initiate the fast phase of spindle elongation in a single transition.

View larger version (81K): [in this window] [in a new window]   Fig. 8. Suppression of cdc28-4 clb5 spindle polarity defects by spc11013. (A) Western blot analysis of paired extracts from independent isolates of asynchronous cdc28-4 clb5 cells expressing HA3-tagged Spc11013p or Spc11013-36A91Ap at permissive temperature. Phosphorylation of this construct (arrowheads) was still dependent on the two CDK consensus sites indicating that other B-type cyclins might still direct phosphorylation of this truncation. Phosphorylation of Spc11013p dependent upon S36 and S91 within CDK sites in otherwise wild-type cells is shown in supplementary material Fig. S4. (B,C) Selected frames from time-lapse sequences for Dyn1p-GFP accumulation at the SPBs and astral MT behaviour during spindle assembly in cdc28-4 clb5 DYN1:GFP cells expressing Spc11013p or Spc11013-36A91Ap. (B) In a cell expressing Spc11013p, Dyn1p-GFP initially marked the old SPB and associated astral MTs (that interacted with the bud cortex). After SPBs separated, the label began to accumulate at the new SPB (0 minutes, arrowhead). Consistent with this intrinsic SPB asymmetry, astral MTs emerging from the new SPB correctly interacted with the mother cortex away from the bud neck. The lag in Dyn1p-GFP acquisition was observed in 60% of cells recorded, n=15 cells. (C) In a cell expressing Spc11013-36A91Ap, both SPBs were marked by the Dyn1p-GFP fusion during separation (0 minutes, arrowheads). In agreement with this lack of intrinsic asymmetry, both SPBs established dynamic astral MT interactions with the bud cortex. A lag in Dyn1p-GFP acquisition was observed in 5% of cells recorded, n=19 cells. Numbers indicate time elapsed in minutes relative to the first frame in which both SPBs were visible with this label. Bars, 2 µm.

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