Figure 2.
mps3 SUN domain mutants are defective in SPB duplication and karyogamy. (A, top) Flow cytometric analysis of DNA content showed that some mps3 SUN domain mutant cells (e.g., mps3-F592S) are haploids (1N and 2N) at the permissive temperature of 23°C, whereas others (e.g., mps3-W487A) spontaneously diploidize (2N and 4N). ts mps3 SUN domain mutants (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200601062/DC1) were grown to midlog phase at 23°C and then shifted to 37°C for 4 h. (middle) Indirect immunofluorescence microscopy revealed that both mutants arrest at 37°C with monopolar spindles: a single microtubule array (anti-Tub1; green) nucleated from one SPB (anti-Tub4; red) associated with one DNA mass (DAPI; blue). Bar, 5 μm. (bottom) Nuclei from large budded cells were examined by EM to further examine SPB number and mitotic spindle structure. Thin section images of representative SPBs, the number cells with the depicted SPB number/structure, and the total number of nuclei examined is shown. Bar, 0.2 μm. (B) The experiment in A was performed with other mps3 SUN domain mutants (Fig. S1). The DNA content of each mutant at 23°C, the percentage of large-budded cells for each sample at 23°C and 37°C, and the percentage of large-budded cells with monopolar spindles at 37°C was determined (n = 200 in three independent experiments). (C) The indicated gene on a 2μ URA3 plasmid (pRS202 or pRS426) was transformed into each mps3 SUN mutant and analyzed for its ability to restore growth at 37°C in a serial dilution assay. 2 OD600 of cells from an overnight culture were spotted onto SD-URA plates in a series of six fivefold dilutions. After 3 d at 37°C, suppression was scored as follows: ++, growth at the fourth dilution and beyond; +, growth at the second or third dilution; −/+, growth only at the first dilution; −, no growth above background. None of the mutants were suppressed by 2μ SPC42, 2μ SPC29, 2μ CNM67, or 2μ NUD1 (not depicted). (D) Levels of wild-type Mps3 or mps3 mutant proteins in cell extracts from A and B were determined by Western blotting with anti-Mps3 antibodies (top). We also included a sample from a strain in which the endogenous copy of MPS3 was replaced with MPS3-GFP (GFP; SLJ911); because Mps3-GFP is 27 kD larger than wild-type Mps3, this allows us to clearly determine the position of endogenous Mps3. G6PDH serves as a loading control (bottom). (E) In the same cells grown at 37°C, localization of wild-type Mps3 and mutant mps3 proteins to the SPB, marked by the end of the microtubule signal, was analyzed by indirect immunofluorescence microscopy with affinity-purified Mps3 polyclonal antibodies. Anti-Mps3 is in red, microtubules are in green, and DNA is in blue. Arrowheads point to SPB-localized Mps3, and the percentage of cells with visible Mps3 at the SPB is indicated (n = 100 in two independent experiments). In addition to what is shown, 38% of mps3-W477A, 58% of mps3-Y502H, 11% of mps3-A540D, and 17% of mps3-Q572L Q573L protein localized to the SPB at the SPB at 37°C; in the remainder of cells, a diffuse staining pattern similar to that of mps3-W487A and mps3-F592S was observed. All of the mutant mps3 proteins localized to the SPB in >80% of cells at 23°C (not depicted). Background staining away from the SPB is due to a combination of Mps3 localization to the nuclear periphery (Jaspersen et al., 2002; Nishikawa et al., 2003) as well as nonspecific cross-reactivity of the antibody; therefore, only Mps3 localization to the spindle poles was analyzed. Bar, 5 μm.