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. 2006 Aug 28;174(5):665–675. doi: 10.1083/jcb.200601062

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Copyright © 2006, The Rockefeller University Press

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Figure 7. — Mps3 localization to the SPB is compromised in mps2-381 mutants. (A) Wild-type (SLJ911) and mps2-381 (SLJ2056) cells in which the endogenous copy of MPS3 was replaced with MPS3-GFP were grown to midlog phase at 23°C and then shifted to 37°C for 3 h. Levels of Mps3-GFP and other SPB components were analyzed by Western blotting with anti-GFP and other polyclonal antibodies. G6PDH serves as a loading control. (B) Mps3-GFP localization to the SPB was examined by epifluorescence microscopy in wild-type cells (arrowheads; top left), and mps2-381 mutants (bottom left) that had been shifted to 37°C. Similarly, indirect immunofluorescence microscopy using anti-Cdc31 antibodies (red) was used to compare Cdc31 staining at the SPB at the ends of microtubules (anti-Tub1; green) in wild-type cells (arrowheads; top right) and mps2-381 mutants (bottom right) at 37°C. In both samples, DNA was visualized by DAPI staining (blue). Bars, 5 μm. (C) Localization of Mps3-GFP and Cdc31 as well as Tub4 and Spc110 was determined in wild-type (SLJ001) and mps2-381 (SLJ2056) cells grown to midlog phase at 23°C and then shifted to 37°C for 3 h as in B, and the percentage of cells where each protein was clearly observed at the SPB was quantitated. Over 100 cells in two independent experiments were analyzed. (D) Strains containing 9xMYC-MPS2 (SLJ1996) or 9xMYC-mps2-381 (SLJ2139) were grown to midlog phase at 23°C and then shifted to 37°C for 3 h. Indirect immunofluorescence microscopyrevealed that both wild-type 9xMYC-Mps2 and 9xMYC-mps2-381 (9E10 anti-MYC; red) localize to the ends of microtubules (anti-Tub1; green) at both 23 and 37°C and colocalize with the SPB protein Tub4 (not depicted). DNA was visualized by DAPI staining (blue). Because mps2-381 cells arrest with monopolar spindles, only a single SPB and Mps2 focus is observed at 37°C in the mutant. Bar, 5 μm.