(A) Cartoon and representative time-lapse inverse fluorescence images of a hydroxyurea (HU)-arrested kar9∆ cell exhibiting typical dynein-mediated spindle movements, analysis of which is presented in panels (B – F). Maximum intensity (X-Y projection; top) and Y-Z projections (bottom) are shown for each time point (NT, neck transit; note, line spans time frames over which the NT occurs). (B – F) Plots of indicated parameters for spindle dynamics in haploid wild-type and mutant strains. Briefly, the mitotic spindles were tracked in 3-dimensions using a custom written Matlab code. Dynein-mediated spindle movements were manually selected from the tracking data, from which velocity (B), displacement (C, per event; or, E, per minute), and the number of dynein-mediated spindle movements per minute (F) were obtained. The fraction of successful neck transits (successful attempts divided by total attempts) were manually scored (i.o., insufficient observations; for H3639P, only one unsuccessful neck transit attempt was observed). Each bar represents the weighted mean ± weighted standard error (or standard error of proportion for D; n = 42 to 60 HU-arrested cells from three independent experiments were analyzed for each strain; diamonds represent mean values obtained from each independent replicate experiment). Statistical significance was determined using an unpaired Welch’s t test (B and E), a Mann-Whitney test (C), or by calculating Z scores (D and F; *, p≤0.1; **, p≤0.05; ***, p≤0.005). Also see Figure 2—figure supplements 1, 2, 3 and 4, and Figure 2—source data 1.