Taxol-induced low tension and kinetochore phosphorylation is insufficient to produce efficient kinetochore detachment without robust microtubule dynamics. (A) Time series depicting sister centromeres (green) and spindle poles (red) for Taxol-sensitive yeast cells (all images have been band-pass filtered in MATLAB). Top: Untreated Taxol-sensitive cells. Bottom: Taxol-sensitive cells treated with Taxol (30 μM). (B) Sister centromere spacing is measured as the spacing between two green lacO-lacI-GFP signals. Sister centromere spacing was plotted against time for each single cell. Left: Sister centromere spacing history for an untreated Taxol-sensitive cell. Right: Sister centromere spacing history for a Taxol-sensitive cell, treated with Taxol. (C) Orange: Combined tension history for untreated Taxol-sensitive cells (n = 15 cells; binned at 1 min intervals; mean ± SEM). Green: Combined tension history for Taxol-sensitive cells, treated with Taxol (n = 19 cells; mean ± SEM). (D) Tension history data was fit to a linear model for untreated (orange) and Taxol-treated (green) cells. The variability in tension from the mean fit line appeared larger for untreated cells (orange open circles) as compared to Taxol-treated cells (green open circles). (E) Cumulative squared error vs. time, with cumulative squared error calculated by squaring the difference between the linear predicted tension value and the actual tension value, at each time point. (F) Tension distribution of untreated (n = 157 cells) and Taxol-treated (n = 103 cells) Taxol-sensitive cells, measured in population sampling experiments. (G) Left: Western blotting to detect phosphorylation of Dam1 in Taxol-sensitive yeast cells. Dam1 detected with α-Dam1 antibody from cell lysates of log phase culture untreated and Taxol-treated cells. Right: Quantification of phosphorylation levels from Western blotting. Each point represents an independent repeat. (H) Fraction of cells with detached kinetochore microtubules, measured in population sampling experiments (untreated n = 157 cells, Taxol-treated n = 103 cells; P = 0.91, Z test). Source data are available for this figure: SourceData F5.
Figure 5.

Taxol-induced low tension and kinetochore phosphorylation is insufficient to produce efficient kinetochore detachment without robust microtubule dynamics. (A) Time series depicting sister centromeres (green) and spindle poles (red) for Taxol-sensitive yeast cells (all images have been band-pass filtered in MATLAB). Top: Untreated Taxol-sensitive cells. Bottom: Taxol-sensitive cells treated with Taxol (30 μM). (B) Sister centromere spacing is measured as the spacing between two green lacO-lacI-GFP signals. Sister centromere spacing was plotted against time for each single cell. Left: Sister centromere spacing history for an untreated Taxol-sensitive cell. Right: Sister centromere spacing history for a Taxol-sensitive cell, treated with Taxol. (C) Orange: Combined tension history for untreated Taxol-sensitive cells (n = 15 cells; binned at 1 min intervals; mean ± SEM). Green: Combined tension history for Taxol-sensitive cells, treated with Taxol (n = 19 cells; mean ± SEM). (D) Tension history data was fit to a linear model for untreated (orange) and Taxol-treated (green) cells. The variability in tension from the mean fit line appeared larger for untreated cells (orange open circles) as compared to Taxol-treated cells (green open circles). (E) Cumulative squared error vs. time, with cumulative squared error calculated by squaring the difference between the linear predicted tension value and the actual tension value, at each time point. (F) Tension distribution of untreated (n = 157 cells) and Taxol-treated (n = 103 cells) Taxol-sensitive cells, measured in population sampling experiments. (G) Left: Western blotting to detect phosphorylation of Dam1 in Taxol-sensitive yeast cells. Dam1 detected with α-Dam1 antibody from cell lysates of log phase culture untreated and Taxol-treated cells. Right: Quantification of phosphorylation levels from Western blotting. Each point represents an independent repeat. (H) Fraction of cells with detached kinetochore microtubules, measured in population sampling experiments (untreated n = 157 cells, Taxol-treated n = 103 cells; P = 0.91, Z test). Source data are available for this figure: SourceData F5.

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