The loss of nonessential RSC subunits causes defective SPB function. (a) Haploid rscΔ spores expressing Spc42-GFP and Hta2-mCherry were freshly germinated and grown to logarithmic phase, and at least 100 large-budded cells were scored for the defects indicated in the micrographs (left). The mean of two technical replicates is plotted. The micrographs are maximum-intensity projections of representative cells containing the indicated SPB defect. Bar, 3 μm. (b) A glucose-repressible HTL1 allele was integrated at the HO locus in a wild-type or htl1Δ strain. Expression of HTL1 was repressed by the addition of glucose, and ploidy was analyzed by flow cytometry at the indicated times. (c) Samples from panel b were imaged, and SPB defects in large-budded cells were quantified as in panel a. At least 100 large-budded cells were scored for SPB defects. 10G, 10 generations. (d) Strains were grown in medium containing glucose for 12 h and then imaged in a microfluidic chamber, for the times specified, in rich medium containing glucose. Corresponding image sequences can be found in Videos 1, 2, 3, 4, and 5. Bar, 3 μm. (e) Maximum-intensity projections of representative HTL1 shut-off cells containing Spc42-GFP after growth in glucose for 12 h, imaged using SIM. Each type of SPB defect is indicated and highlighted by a colored arrow. The insets show higher-magnification images of the SPBs that are highlighted by arrows. Bar, 3 μm. The corresponding flow cytometric analysis is shown in Fig. S2 d.