Profiling the temperature sensitivity of ts strains. (a) Left panel; general interpretation of high-resolution liquid growth profiles of strains harboring ts alleles. Growth rate (population doubling time) was determined by measuring the slope of the exponential phase of the growth curve. Lag was given by the intercept of the initial density and the slope, and growth efficiency was calculated as the total change in density for cells having reached stationary phase. Right panel; sample growth curves of the strains with the act1-105 ts allele, obtained by micro-cultivation over a temperature range. (b) The fraction of all ts strains that exhibit significant (P < 0.001) temperature sensitive defects in growth rate (blue), lag (green) and/or efficiency (red) phases compared to wild type (WT) at the indicated temperatures. (c) Uncentered hierarchical clustering of ts allele growth profiles over all three growth variables. Profile similarity was measured using a Pearson similarity metric and average linkage mapping. The ts-strain sensitivity ratio is expressed as log2(WT/ts strain) at temperature X − log2(WT/ts strains) at 22 °C. ER, endoplasmic reticulum.

Zaragozic acid rescues bet2-1 and cdc43-2 ts phenotype. (a) Isoprenoid pathway in S. cerevisiae. The solid lines indicate one step and the dashed lines indicate multiple synthetic steps in the ergosterol and protein prenylation pathways^44–46. (b) A pool of 440 barcoded ts mutants was grown in rich (YPD) medium in the presence of 4 mM of zaragozic acid A (ZA) or DMSO at 36.5 °C. Genomic DNA was prepared from cells after five generations of growth. Molecular barcodes were amplified by PCR and hybridized to a microarray (GeneChip Genflex Tag 16K Array v2, Affymetrix). The x axis represents the 440 ts strains ordered alphabetically by systematic name. The y axis represents the log2 ratio of barcode hybridization intensity between the ZA treatment and the solvent (DMSO) treatment. Mutants with highest log2 ratios at the restrictive temperature were identified as suppressors. (c) Yeast cells were grown in YPD with or without ZA in a 96-well plate at 36.5 °C. Fitness was defined as the ratio of doubling times measured in DMSO compared to ZA treatment. Error bars represent s.d. for four independent experiments.

High-content screening of the ts allele collection identifies abnormal spindle morphology associated with cohesin and condensin mutants. (a) Fluorescent reporters introduced into the ts collection. The left panels show the localization of six different GFP/RFP-tagged marker proteins expressed in wild-type cells as well as the corresponding computationally processed images. Representative mutants with defects in localization of particular markers are shown on the right. Markers included a plasma membrane marker (Psr1p-GFP), a reporter of DNA damage (Ddc2p-GFP), a nuclear marker (Mad1p-NLS-RFP), a mitotic spindle reporter (GFP-Tub1), a mitochondrial marker (OM45p-GFP) and an actin reporter (Sac6p-GFP). (b) A sub-population of cohesin and condensin mutants exhibit abnormal spindle morphology. Spindle length and morphology were quantified in large-budded wild-type cells and strains harboring a cohesin (smc3-1) or condensin (smc2-1) mutant allele grown asynchronously at permissive (26 °C) and restrictive temperatures (32 °C). The fraction of cells exhibiting either short (upper panel) or fishhook-shaped spindles (lower panel) were quantified in three independent populations; error bars represent s.d. across three independent experiments. (c) Ipl1p-GFP localization in cohesin (smc3-1) and condensin (smc2-8) mutants. Ipl1p-GFP localization to the mitotic spindle was monitored in wild-type, smc3-1 and smc2-8 mutants at permissive (26 °C) and restrictive temperatures (32 °C). Representative cells with short (top panels) and long (bottom panels) spindles are shown. White arrows indicate Ipl1p-GFP localization to the kinetochore in a cohesin mutant at the restrictive temperature. The fluorescent intensity line profile of Ipl1p-GFP for a representative cell is also shown. (d) Wild-type and mutant cells were divided into three separate groups based on spindle length (2–4 µm, 4–6 µm, >6 µm) as measured by assessment of an RFP-Tub1p marker. The percentage of cells in each spindle length group that exhibited mis-localized Ipl1p-GFP was measured at permissive and restrictive temperatures. Error bars represent the s.d. across three independent experiments.

Relationship between cohesin, condensin and chromosomal passenger complexes (CPC). (a) Physical interaction network illustrating protein-protein interactions involving cohesin, condensin and CPC complexes. Nodes represent individual proteins and edges represent physical interactions. (b) Cohesin, condensin and CPC complex localization. Ipl1p-GFP, Smc3p-GFP and Smc4p-GFP localization was assessed with respect to the spindle pole marker, Spc29p-RFP in a wild-type strain. Fluorescent micrograph images illustrate representative single cells.