(A) Effects of benomyl on chromosome biorientation. Left: Kinetics of chromosome IV biorientation visualized using a centromere-proximal TetO array. Micrographs depict representative images of cells showing fluorescently labeled TetR-GFP bound to the TetO array and Spindle Pole Bodies (SPB). Scale bar ~3.2 µm. Second from the left: Quantification of the fraction of cells with bioriented chromosome IV. Note that the reduced fraction of untreated cells with bioriented chromosomes 105 min after release from G1 is because most cells complete anaphase by this time. (n = 259, 252 and 398 at 45, 75 and 105 min respectively in normal media, one repeat. n = 855, 816 and 626 respectively at 45, 75 and 105 min in benomyl-containing media from three technical repeats). When the two CENIV foci were unresolvable and localized in the vicinity of one of the two SPBs, they were scored as monopolar attachments. When the two CENIV foci were clearly separated from each other and located along the spindle axies, they were considered to be bioriented. Middle: Representative micrographs of cells grown in benomyl media containing unattached kinetochores recruiting Bub1. Arrow heads indicate the unattached kinetochores with Bub1 localizations. Scale bar ~3.2 µm. Right: Quantification of the number and frequency distribution of unattached kinetochores in yeast cells growing in media containing benomyl. The unattached kinetochore number was estimated by comparing the Spc105-GFP fluorescence from the unattached kinetochore cluster (marked by Bub1-mCherry recruitment) with the total fluorescence from the kinetochores along the spindle axis. (n = 686, 501 and 543 at 45, 75 and 105 min respectively, pooled from two technical repeats). Red dashed line in relative frequency plot indicates the average number of unattached kinetochores observed after 90 min nocodazole treatment of wild type yeast cell (Aravamudhan et al., 2016). (B) Left: Micrograph displays representative cells in metaphase and anaphase in benomyl-containing media. Scale bar ~3.2 µm. Right: Quantification of the number of anaphase cells observed after the indicated time following release from a G1 arrest for the indicated strains. (mean+ s.e.m, n = 1029, 889 and 992 at 45, 75 and 105 min respectively for WT cells, pooled from three experimental repeats. n = 890, 864 and 812 at 45, 75 and 105 min time points respectively for #2,6, pooled from three technical replicates). (C) Top: Schematic of the Spc105 phosphodomain with the amino acid sequence indicated at the top. Bottom: Quantification of the evolution of cell density of the indicated strains in rich media (left) and media-containing benomyl (right). (D–E) Assessment of the sensitivity of yeast strains to benomyl using the spotting assay. Schematics on the left show the active motif number and its position. The blue bar represents the ‘basic patch’ in Spc105, which promotes PP1 recruitment. The photographs on the right show the results of spotting a serial dilution of yeast cells on rich media (YPD) and media containing benomyl (20 or 30 μg/ml). (F) Assessment of chromosome loss by colony sectoring assay. Left: Images of WT, mad1Δ and #4–6 colonies grown in YPD plates. Right: Bar graph shows the rate of loss of SUP11 containing chromosome fragment measured as the number of red/half red colonies (example shown on the right) for every 1000 colonies plated. N = 5081, 4406 and 4965 for WT, mad1Δ and #4–6 respectively, pooled from at least six technical repeats.