(A) Schematic representation of the middle domain swaps CFLFFC and FCSCCF. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and either CFLFFC-EGFP or FCSCCF-EGFP. (C–F) Graphs showing quantification of stage 1 neurons comparing the effects of middle domain swaps on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F). CIP4S-EGFP (n = 23 cells), CFLFFC-EGFP (n = 17 cells), FBP17L-EGFP (n = 24 cells), or FCSCCF-EGFP (n = 17 cells). (G) Stacked bar graph comparing the percentage of neurons in stage (st) 1, 2, and 3 for neurons expressing CIP4S-EGFP (n = 50) versus CFLFFC-EGFP (n = 54) and FBP17L-EGFP (n = 61) versus FCSCCF-EGFP (n = 52) at 12 h postplating. One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images of neurons and 1 µm in insets.