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. 2018 Dec 15;29(26):3105–3118. doi: 10.1091/mbc.E18-02-0115

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© 2018 Bärenz, Kschonsak, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

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FIGURE 4: — MT EB3-tracking measurements reveal a significant decrease in spindle symmetry in the absence of Ccdc61. (A–C) HeLa cells stably expressing EGFP-EB3 were used for EB3-tracking experiments. Cells were either treated with control (ct), Ccdc61 O2 siRNA (O2), or a combination of Ccdc61 O2 siRNA together with FLAG-Ccdc61 siRNA-resistant construct (O2R). To study mitotic properties, like directionality and orientation, the individual EB3 tips were imaged every 0.13 s and analyzed via an automated process. (A) Example images of a single time frame (top row) and a time projection from 125 s recordings (bottom row) of HeLa cells treated as indicated. Western blot shows the efficiency of indicated Ccdc61 Oligo that was tested by its ability to deplete transiently transfected FLAG-Ccdc61 or siRNA-resistant FLAG-Ccdc61 (O2R). α-tubulin served as a loading control. (B) EGFP-EB3 tracks from HeLa cells were analyzed as described in the Materials and Methods section and as published in the preprint (Jafarpour and Lorenz, 2017). The table represents velocity (µm/min), displacement (µm), duration (s) (mean ± SD.) and the total number of analyzed tracks (of 10 independent cells). The significance was calculated for the three conditions by a one-way ANOVA, and the alpha value at which there was no significant difference (n.s.d.) within the group as well as the p value is provided. (C) Shown are the results of calculated asymmetry and directionality indices of the indicated treated HeLa cells with 10 image series per group based on the histogram of calculated orientations (Supplemental Figure S4, B and C). Each mitosis event is characterized with two figures of merit, resulting in a single point. The y-axis represents the degree of asymmetry, whereby a low value indicates a symmetric direction of MT growth (low asymmetry) and a high value indicates an asymmetric direction of MT growth (reduced symmetry). The horizontal axis represents the directionality of MT growth, indicating different levels of point symmetry in MT growth paths.