Short SKAP displays potent EB motif–dependent microtubule plus-end tracking activity in mitotic cells. (A) Still images from spinning disc confocal videos of short SKAP-GFP (see Video 2). (B, top) Diagram of the SKAP ΔEB mutation. (bottom) Individual fluorescence sections from live cell imaging of mitotic SKAP and SKAP ΔEB mutant GFP-tagged cell lines. (C) Immunofluorescence localization of EB1 and SKAP. (top) SKAP overlaps with the trail of the EB comet, but does not colocalize with the tip. Inset shows a zoomed-in and channel separated view of the boxed region. (middle) Mitotic cells exhibiting wild-type (WT) SKAP plus-end tracking and the ΔEB mutant. (bottom) Maximum-intensity linescans of the microtubule plus ends marked with an arrow above. Intensities are plotted as a percentage of the maximum intensity quantified in this image set. (D) Individual fluorescence sections showing SKAP localization for the depletion and replacement experiments. DNA is scaled independently for each image. (E) Quantification of mitotic chromosome alignment for SKAP depletion and rescue experiments. Cells were quantified by observing DNA and spindle morphology (as in Fig. 2 C). (F) Mitotic timing (nuclear envelope breakdown [NEBD]-anaphase) quantified from videos (as in Fig. 3 E). n = 52 cells/condition. Based on a two-tailed t test, the datasets are statistically different (P = .0065). WT rescue data are duplicated from Fig. 3 E for comparison. Bars, 5 µm.