Figure 4

Pre-anaphase spindle alignment is perturbed in γtub-Y362E cells. (a) Schematic depicting the method used to compute the distance between proximal pole and bud neck (spindle positioning) and spindle alignment in relation to “perfect alignment”. Perfect alignment is defined as a spindle with a 1D projected spindle length ~3D spindle length. (b) Distance of the proximal pole from the bud neck; error bars show standard deviation. *Indicates statistical difference with p < 0.0001; n.s. denotes no significant difference. (c) Heatmaps showing the projected 1D spindle length as a function of true 3D spindle length in wild-type (WT), γtub-Y362E, dyn1∆ and kar9∆ cells. Color bar shows the frequency of time points normalized to the total number of time points of all cells. Incorrect alignment occurs when the new pole is proximal to the bud. (d) Difference map comparing WT (shown in (c); upper left) to γtub-Y362E (top), dyn1∆ (middle) and kar9∆ (bottom). Green indicates areas where the frequency of the WT was increased relative to the mutant, red indicates areas where the frequency of the mutant was increased relative to WT. Instantaneous projected 1D spindle length plotted as a function of true 3D spindle length of representative (e) WT and (f) γtub-Y362E cells. Time is indicated by the color gradation from dark (start) to light (end). (g) The γtub-Y362E mutation increases the range of unaligned spindle orientations and incorrect alignment when combined with kar9∆ and dyn1∆ mutants. Heatmaps showing the projected 1D spindle length in double mutants γtub-Y362E; dyn1∆ (upper left) and γtub-Y362E; kar9∆ cells (lower left). Plots showing the differences between the single mutants and double mutants. Green indicates areas where the frequencies of the single mutant were larger than the double mutants, red indicates areas where the frequencies of the double mutant were increased.