Aberrant equatorial centering and dynein distribution after MARK2 depletion can be rescued by MCAK depletion. (a) Immunoblots show depletion extent of MARK2 and MCAK in lysates of HeLa^{His2B-GFP;mCherry-Tub} cells after siRNA treatment with siRNA oligonucleotides as indicated. γ-Tubulin is used as loading control. (b) Representative time-lapse images of HeLa^{His2B-GFP;mCherry-Tub} cells treated with siRNA against MARK2 or MCAK alone or both MCAK and MARK2. Equatorially centered (C) and off-centered (OC) spindles are marked. mCherry-tubulin signal alone is shown and the signal was used to outline the cell cortex (circles). Bars, 2 μm. (c) Graph of percentage of equatorially centered spindles assessed from time-lapse videos as in b. Error bars are SEM values across three experimental repeats. (d) Temporal evolution of spindle positions in five representative cells treated with indicated siRNA showing normal recentering of equatorially off-centered spindles in cells treated with siRNA against either MCAK alone or both MCAK and MARK2 but not MARK2 alone. A, anaphase. Red and green boxes indicate equatorially off-centered and centered spindles, respectively. (e) Representative live-cell images of HeLa^DHC-GFP cells treated with the indicated siRNA oligonucleotides. Yellow arrowheads mark cortical DHC-GFP localization. Bars, 15 μm. (f) Bar graph shows percentage of HeLa^DHC-GFP cells with crescent or diffused cortical dynein localization after treatments with the indicated siRNA oligonucleotides. Error bars are SEM values across data collated over three independent experiments. (g) Successful spindle rotation requires equatorial centering: Cartoon shows two separate pathways governing equatorial and longitudinal spindle positions: (i) LGN directly controls cortical dynein recruitment and, in turn, cortical dynein–mediated pull that powers spindle displacement (blue arrows). (ii) In contrast, MARK2/Par1b controls astral microtubule length and dynamics and ensures equatorial spindle centering (orange arrows). Both equatorial centering mediated by MARK2 and cortical pulling powered by dynein are needed for successful spindle rotation. Equatorial off centering and longitudinal oscillations have an indirect impact on cortical dynein crescent formation.