Figure 4.—

The mad2 clb3 clb4 strain has defects in SPB separation and spindle assembly. (A) A conditional mutant of the mad2 clb3 clb4 strain was generated. The mad2 clb3 clb4 GALL-CLB3 GAL4-HER or wild-type strain was plated on YPD, YPD plus 0.5× estradiol, YPD plus 1× estradiol, and YPD plus 2× estradiol. The plates were grown at 30° for 2 days to test viability. (B) The mad2 clb3 clb4 GALL-CLB3 GAL4-HER TUB1-CFP or TUB1-CFP strain was incubated in YPD with estradiol first, and the estradiol was removed for 6 hr to turn off CLB3. The cells were then briefly fixed with formaldehyde and stained with DAPI. They were observed under a fluorescent microscope with a cyan fluorescent protein (red image) and DAPI (blue image) channel. The images were merged and contrast enhanced using MatLab software to visualize the fluorescent signals for reproduction. These manipulations improved image reproducibility when printed but had no effect on the qualitative interpretation (original images available upon request). (C) The cells were categorized into four groups in which cells have no spindles, spindles as a dot, short spindles, or elongated spindles. At least 100 cells were counted for each sample, and the percentage of each category was calculated. Note that this scoring was performed with the unenhanced images. (D) The mad2 clb3 clb4 GALL-CLB3 GAL4-HER SPC110-GFP or CLB3 CLB4 SPC110-GFP strain was grown in YPD with estradiol, and then estradiol was removed for 6 hr. The cells were observed under a fluorescent microscope to visualize Spc110-GFP. The fluorescent image and DIC image were merged. (E) Cell images such as those in D were counted and categorized into four groups (from left to right below the x-axis): single cells with one SPB; small-budded cells with one SPB; small-budded cells with two SPB; large-budded cells with two separated SPB in the mother and daughter cells; and large-budded cells with one SPB.