Figure 2. mps1-8 cells fail in SPB duplication when grown at their restrictive temperature (36 C). (A and B) Immunofluorescence images of a mps1-8 (ACY71-14b) strain grown at the permissive (A, 25 C) or restrictive (B, 36 C) temperature for 3 h after release from an -factor–induced G1 arrest. These cells were fixed and stained with DAPI to visualize DNA (blue), an antibody against -tubulin to visualize microtubules (red), and Spc42-GFP in this strain (ACY71-14b) identifies the SPBs (green). (A) In mps1-8 cells grown at the permissive temperature (25 C), two foci of Spc42-GFP in the large budded cell indicates that the SPB has duplicated. In this image, a short spindle can be seen between the two SPBs. (B) At the restrictive temperature (36 C), only a single foci of GFP signal is observed in large budded cells (92%, n 42), indicating SPB duplication has failed to occur. (C–E) Electron micrographs of asynchronously growing mps1-8 (ACY66-4) and mps1-1 (WX241-3b) diploids shifted to the restrictive temperature for 5 h. An unduplicated SPB was detected in serial sections of large budded mps1-8 and mps1-1 cells. Only one section is shown here. The unduplicated SPB of mps1-8 cells (D; n 17) is associated with half-bridge (HB, arrow) material but lacks the extended half-bridge typical of mps1-1 mutants (E, HB, arrow). NPC, nuclear pore complex. Bars: (A and B) 2.0 m; (C) 0.4 m; (D) 0.2 m; (E) 0.1 m. terials and methods) (Geiser et al., 1997). These alleles will be described elsewhere. Using temperature sensitivity as a screen for potential SPB duplication defects, we isolated mps1-8 (Fig. 1 A) and showed it is a recessive mutation (un- published data). Sequencing revealed that mps1-8 contained multiple mu- tations (Fig. 1 B); none of the 10 mutations that resulted Mps1p regulates assembly of Spc42p | Castillo et al. 455 in amino acid changes individually conferred conditional growth (unpublished data). It is likely that some combina- tion of these mutations is responsible for the mps1-8 pheno- type, and all further analysis was performed using the origi- nal mps1-8 allele. mps1-8 mutants fail in SPB duplication We have established previously that the essential MPS1 cel- lular function is in SPB duplication (Weiss and Winey, 1996). To test if the mps1-8 conditional growth defect might reflect this role, we monitored SPB duplication in mps1-8 cells using immunofluorescence microscopy and EM. Asynchronously growing mps1-8 cells were arrested in G1 using the mating pheromone -factor, released to the permissive (25 C) or restrictive (36 C) temperature for 3h, and processed for indirect immunofluorescence. The major- ity of mps1-8 cells complete SPB (shown in green) duplica- tion at 25 C as expected, but they fail to duplicate their SPB at 36 C (92%, n 42; Fig. 2, A and B). Instead, these cells show a typical S. cerevisiae mitotic arrest state with large buds in which the single unduplicated SPB is associated with cellular DNA (blue) and a focus of microtubules (red) (Fig. 2 B). By immunofluorescence, the SPB duplication defect in the mps1-8 strain is identical to the SPB defect observed in previously characterized conditional MPS1 mutants (Schutz and Winey, 1998). The morphology of the SPB in mps1-8 cells grown at the restrictive temperature was further examined using EM (see Materials and methods). Serial sections of large budded cells were examined, and a single SPB was found in all cases, veri- fying that SPB duplication fails in mps1-8 cells grown at the restrictive temperature. As in other MPS1 mutants, the undu- plicated SPB in mps1-8 cells is associated with half-bridge material, but unlike them this structure is not elongated in mps1-8 cells (Fig. 2, C–E) (Winey et al., 1991; Schutz and Winey, 1998). It is not yet clear if the subtle difference in the terminal SPB phenotype of the mps1-8 mutant and other MPS1 mutants indicates they are failing at different points in SPB duplication. mps1-8 mutants are competent to activate the spindle checkpoint As mentioned, the previously characterized conditional MPS1 mutants are defective in both SPB duplication and the spindle checkpoint (Winey et al., 1991; Weiss and Winey, 1996; Schutz and Winey, 1998). We show that the mps1-8 mutant fails in SPB duplication at the restrictive temperature. To determine if mps1-8 cells also fail in the spindle checkpoint, we compared the mps1-8 strain with mps1-1, a strain known to be defective in activating the spin- dle checkpoint, and mps2-1, a strain able to activate the spindle checkpoint with a monopolar spindle (Hardwick et al., 1996; Weiss and Winey, 1996). Asynchronously grow- ing cultures of mps1-8, mps1-1, and mps2-1 cells were syn- chronized in G1 with -factor, released at 25 or 36 C, and samples for flow cytometry and budding indices were taken after 2 and 3 h. At the restrictive temperature, mps1-8 cells exhibit a mitotic arrest similar to that observed for mps2-1 cells; the majority of the cells have a large budded cell mor- phology, a G2 DNA content (Fig. 3, A and B), and accumu-