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. 2019 Jan 7;218(1):350–379. doi: 10.1083/jcb.201711023

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© 2018 Bagonis et al.

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Figure 8. — ARP2/3 inhibition via CK666 treatment induces GC morphological changes and stagnation of neurite outgrowth. (A) GCA segmentation of N1E-115 GCs on laminin before (left) and after (right) treatment with DMSO or 25 µM CK666. Bar, 10 µm. (B) CK666 treatment increases filopodia length (top) and decreases the percent length each actin bundle is embedded in veil (bottom). (i) Percent change in the median of the actin bundle distribution (scatter points) before and after treatment. n = 3 GC movies per treatment type. Lines, mean of distribution per treatment condition; dark shaded regions, 95% CIs about the means. *, P = 0.1: two-tailed permutation t test of the means. (ii) Distributions of actin bundle measurements. Boxplot: 25th, 50th, and 75th distribution quantiles. Whiskers, 1.5 × (Q_75% − Q_25%). ***, P < 0.001: two-tailed permutation test of the medians. (C) ARP2/3 inhibition via CK666 treatment induces functional transitions from neurite elongation to neurite pausing/retraction. (i) Summary of the percent time individual neurites were paused or retracting before and after treatment. n = 3 GC movies per condition. (ii) Neurite outgrowth lengths/velocities colored by neurite outgrowth state. Dashed line: threshold for pausing. (D) Visualization of morphological heterogeneity among individual GC movies. (i) GCA features calculated per frame for D-reduction. (ii) MDS plot of morphology features (72 GC movies: 8,454 frames). Probability density isocontours for entire dataset visualized in 3D (top) and 2D (bottom). (iii) Example of full bagplot (i.e., 2D boxplot; Rousseeuw et al., 1999) for a single 5-min trajectory before acute perturbation. Dark shaded contour: bag; light shaded contour: fence; circles: individual data points. Black star: outlier data point. Cross: Tukey median. (iv) Bagplot “bags” for six GC movies before acute treatment with DMSO (black) or 25 µM CK666 (blue). (E) CK666 treatment induces a shift in morphology space, coupled to neurite outgrowth state. Two GC movies acutely treated with DMSO (left) or 25 µM CK666 (right). Per-frame coordinates in reduced morphology space colored by neurite outgrowth state (i) or timing relative to treatment (ii). Each movie: 10 min total, 5-s intervals.