MARK2 is required for equatorial spindle centering up to anaphase. (a) Representative single-plane deconvolved images of z stack videos of HeLaHis2B-GFP;mCherry-tubulin cells treated with MARK2 or control siRNA oligonucleotides as indicated. Only mCherry-tubulin signals are shown for clarity. Bars, 5 µm. Arrowheads point to off-centered spindles. Circles highlight the cell cortex. Note that the cell showed in MARK2-1 row is shown in Fig 1 e. (b) Graph of the percentage of mitotic cells with equatorially centered spindles at 8 or 12 min after NEBD. (c) Uncropped immunoblot showing the expression of siRNA-resistant MARK2-siRes-GFP in cells treated with control or MARK2 siRNA. Lysates of HeLaMARK2-siRes-GFP cells treated with siRNA and exposed to tetracycline as indicated were immunoblotted with antibody against hMARK2; anti–γ-tubulin antibody was used as loading control. Endogenous MARK2 and Tet-inducible siRNA-resistant MARK fused with YFP are shown with arrows. (d) Bar graph showing the percentage of mitotic HeLaMARK2-siRes-GFP cells with equatorially centered spindles. Control or MARK2-1 siRNA–treated HeLaMARK2-siRes-GFP cells in the presence or absence of tetracycline (as indicated) were treated with MG132 to arrest them at metaphase prior during live-cell imaging. Cell expressing (green; +) or not expressing (nongreen, –) the siRNA-resistant form of MARK2-siRes-GFP were imaged, and chromosome positions were ascertained using differential interference contrast, which was used to determine equatorially centered or off-centered metaphase plates. (e) Bar graph comparing the extent of equatorial centering of spindles at 8 min after NEBD versus anaphase onset in control or MARK2-1 siRNA–treated cells as assessed from time-lapse videos as shown in a. Error bars represent SEM across three experimental repeats. P-values were obtained using a proportion test on percentage values * and # indicate significant and insignificant difference, respectively.
