1. Microtubule perturbations cause Ste5 patches to form less reliably, delay patch formation, and cause patches to persist for less time
Microtubule perturbations cause Ste5 patches to form less reliably, delay patch formation, and cause patches to persist for less time We stimulated reference (“WT”), Tub1‐828‐expressing, Δbim1 and Δgim4 derivatives of MW003 (bearing three copies of PSTE5‐STE5‐3xYFP, Ventura et al, 2014) with 1 μM pheromone and imaged them over time for up to 3.5 h. Single‐cell measurements of Ste5 patch dynamics. Arrows indicate first and second Ste5 patches. Images show examples of a cell with two co‐existing Ste5 patches (top) and a cell with an interval without detectable Ste5 patches between the first and second patch (bottom). The lines below mark the dynamic features we quantified: time to 1st patch (polarization), duration of 1st and 2nd polarization, interval in which 1st and 2nd polarization overlap, or gap between them, and time from 1st to 2nd polarization. Scale bar: 2 μm.Bar graph plots show qualitative defects observed: cells with no polarization, cells with gaps between first and second polarizations and cells with overlapping (coexisting) polarizations. Error bars represent the standard error as calculated by bootstrapping (104 resamples), and asterisks indicate significant difference from WT as calculated by Fisher's exact test for count data. Table shows quantitative defects. Data correspond to the mean ± SEM, the standard deviation ± SE and the coefficient of variation (standard deviation divided by the mean) ± SE. Standard errors were calculated by bootstrapping (104 resamples), and significant differences from WT were calculated by permutation tests (104 permutations). Values for the probability P of the observed data under the null hypothesis that each mutant strain is no different from WT (WT1 vs. TUB1‐828, and WT2 vs. Δbim1 and Δgim4) are shown by asterisks: *P < 0.05; **P < 0.01; ***P <  Experiments were done in three biological replicates (N = 3). As no important differences were observed among replicates, cells were pooled for the analysis. At least 80 cells of each strain were quantified (N > 80).
C Gustavo Pesce et al. Mol Syst Biol 2018;14:e7390
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