Mispositioned metaphase spindles in SKAP ΔEB mutant cells are accompanied by changes in astral microtubule behavior. (A) Individual sections showing IF localization of p150Glued as a marker for dynein/dynactin from cells in which wild-type (WT) SKAP or the SKAP ΔEB mutant replaces endogenous SKAP. Accumulation of cortical dynactin is marked with arrows. We were unable to find an example of aberrantly localized dynactin (i.e., higher on the cortex closer to the shifted spindle) in two independent experiments. (B) IF images (deconvolved sections) of mouse anti-EB1 localization in SKAP WT and SKAP ΔEB cells. (far right) EB1 tracks from the SKAP ΔEB mutant cell showing growth along cortex. Also see Fig. S3 D. (C) IF images (deconvolved sections) showing microtubule and rabbit anti-EB1 localization. Boxed regions are enlarged on right (∼2×) to show plus ends of microtubules growing laterally along the cortex. Images in this panel are scaled individually for clarity. (D) Montages generated from time-lapse videos of EB3-tdTomato in SKAP WT and SKAP ΔEB cells. Top images are the first frame of the video, with the red boxed region used to create the montage. EB3 comets that exhibit clear lateral growth along cortex are marked with arrows. For the SKAP ΔEB cell, Fiji stack registration was used to stabilize the spindle, which exhibited rocking during the course of the video. See Video 5. Bars: 5 µm; (inset/zoom) 1 µm.
