The budding yeast RSC complex maintains ploidy by promoting spindle pole body insertion

Sing et al. characterize an unanticipated role for the Saccharomyces cerevisiae RSC complex in ploidy maintenance. They show that RSC promotes the distribution of Nbp1 and Ndc1 to the spindle pole body (SPB) to facilitate SPB maturation and accurate chromosome segregation.

. Levels of Nbp1 and Ndc1 decrease at SPBs when Htl1 is depleted. (a) A wild-type or HTL1 shut-off strain expressing NBP1-GFP and a highcopy-number plasmid expressing NBP1 was grown in medium containing glucose for the indicated time, and ploidy was monitored by using flow cytometry. Histograms correspond to the images in Fig. 7 c and the quantification in Fig. 7 (b and d) as well as panels c and d. (b) Raw Nbp1-GFP levels at >200 SPBs in a wild-type or HTL1 shut-off strain are plotted and correspond to the quantification in Fig. 7 d. (c) Raw Spc42-mCherry levels at >200 SPBs in a wild-type or HTL1 shut-off strain are plotted and correspond to the quantification in Fig. 7 d. (d) A wild-type or HTL1 shut-off strain expressing NDC1-GFP was grown in medium containing glucose for the indicated time, and ploidy was monitored by using flow cytometry. Histograms correspond to the quantification in Fig. 7 e. (e) Raw Ndc1-GFP levels at >200 SPBs in an HTL1 shut-off strain are plotted and correspond to the quantification in Fig. 7 e. (f) Raw Spc42-mCherry levels at >200 SPBs in an HTL1 shut-off strain are plotted and correspond to the quantification in Fig. 7 e.
Tables S1, S2, and S3 are provided as Excel files. Table S1 is a list of all diploidized rsc-specific morphological traits. Table S2 shows transcript changes in rsc mutants compared with wild type by RNA sequencing. Table S3 is an SGA analysis of rscΔ strains.    Video 1. Time-lapse confocal images of normal SPB function in a wild-type strain. A wild-type strain expressing Spc42-GFP and Hta2-mCherry was grown to logarithmic phase in medium containing galactose and then grown in medium containing glucose for 12 h before being loaded into a microfluidics plate. Continuous flow of medium containing glucose was provided to trapped cells for 8 h. Images were acquired every 30 min at room temperature, and the resulting 17 frames were displayed at 2 frames per second. Corresponding images can be found in Fig. 3 d. Bar, 3 µm.

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Video 2. Time-lapse confocal images of adjacent SPBs in an HTL1 shut-off strain. An HTL1 shut-off strain expressing Spc42-GFP and Hta2-mCherry was grown to logarithmic phase in medium containing galactose and then grown in medium containing glucose for 12 h before being loaded into a microfluidics plate. Continuous flow of medium containing glucose was provided to trapped cells for 8 h. Images were acquired every 30 min at room temperature, and the resulting 17 frames were displayed at 2 frames per second. Corresponding images can be found in Fig. 3 d. Bar, 3 µm.

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Video 3. Time-lapse confocal images of a dead SPB in an HTL1 shut-off strain. An HTL1 shut-off strain expressing Spc42-GFP and Hta2-mCherry was grown to logarithmic phase in medium containing galactose and then grown in medium containing glucose for 12 h before being loaded into a microfluidics plate. Continuous flow of medium containing glucose was provided to trapped cells for 8 h. Images were acquired every 30 min at room temperature, and the resulting 17 frames were displayed at 2 frames per second. Corresponding images can be found in Fig. 3 d. Bar, 3 µm.
Video 4. Time-lapse confocal images of unseparated SPBs in an HTL1 shut-off strain. An HTL1 shut-off strain expressing Spc42-GFP and Hta2-mCherry was grown to logarithmic phase in medium containing galactose and then grown in medium containing glucose for 12 h before being loaded into a microfluidics plate. Continuous flow of medium containing glucose was provided to trapped cells for 8 h. Images were acquired every 30 min at room temperature, and the resulting 17 frames were displayed at 2 frames per second. Corresponding images can be found in Fig. 3 d. Bar, 3 µm.
Video 5. Time-lapse confocal images of multiple SPBs in an rsc2Δ strain. An rsc2Δ strain expressing Spc42-GFP and Hta2-mCherry was grown to logarithmic phase in medium containing glucose and loaded into a microfluidics plate. Continuous flow of medium containing glucose was provided to trapped cells for 8 h. Images were acquired every 30 min at room temperature, and the resulting 17 frames were displayed at 2 frames per second. Corresponding images can be found in Fig. 3 d. Bar, 3 µm.