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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 7. — Morphodynamic analyses of select Rho GTPase pathway KDs. (See Fig. 2 A). (A) GC scale morphodynamic profiles extracted by GCA. Left: Example z-score calculation; scatter plots: mean filopodia length per 10-min GC movie (1,995–9,944 filopodia per movie). Midline bars: Mean of treatment distribution (3–20 GC movies per condition); dark colored regions: 95% CI of the mean; light colored regions: ±SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001: two-sided permutation t test of the means. Right: Hierarchical clustering of siRNA z-score vectors. Above clustering: z-scores for 10-min neurite outgrowth rate (a global/functional feature). (B) N1E-115 Rho GTPase perturbation dataset (n = 66 GC movies) separated into high (i) and low (ii) net neurite outgrowth clusters for further analysis in C. Insets: Prototypical neurite length trajectories for high (right) and low (left) neurite outgrowth clusters. (C) Robustly elongating Cdc42 KD GCs exhibit enhanced filopodia lengths with no change in veil/stem thickness. Select GC features for high (3–10 GC movies per condition; 2,743–10,216 actin bundles per movie; i) and low (2–10 GC movies per condition; 2,146–8,280 actin bundles per movie; ii) outgrowth neurites. Scatter plots as defined in A. *, P < 0.05; **, P < 0.01; NS, P > 0.05: two-sided permutation t test of the means. Insets: Example images, respective cluster. (D) Positive correlation between filopodia length and maximum filopodia curvature. (i) Scatter points: Spearman r between the two features per GC movie; 5–20 GC movies per treatment; 1,299–7,060 filopodia per movie. (ii–vi) Example correlation plots. Scatter points: single filopodium. N filopodia: (ii) 4,952; (iii) 3,739; (iv) 4,647; (v) 3,740; (vi) 2,328. ***, P < 0.001: Benjamini–Hochberg adjusted. (E) Enhancement of filopodia curvature observed upon Rho GTPase perturbation (top) is primarily a secondary effect of increased filopodia length (bottom). 5–20 GC movies per treatment; 1,299–7,060 filopodia per movie (top); 25–519 filopodia per movie (bottom). *, P < 0.05; **, P < 0.01; ***, P < 0.001: two-sided permutation t test of the means. See Videos 5 and 6.