Actα promotes formation of axonal collateral branches in motoneurons. (A) Motoneurons were transduced with lentivirus expressing shRNA targeting 3′ UTRs of actin isoforms. Immunostaining with phalloidin and against Tau and tubulin labels axons and mature branches. GFP is coexpressed with shRNAs to label transduced cells. (B) In Actα-depleted neurons, the number of axons without collateral branches increases, and the number of axons with three or more branches decreases (**, P < 0.01 for n = 5). (C) Knockdown of Actβ increases number of axonal collateral branches (*, P < 0.031; **, P < 0.01 for n = 6). (D) Depletion of Actγ does not affect branch formation (n = 4). Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test. (E) Dendrites are immunolabeled against Tau and Map2. (F–H) Motoneuron viability is not affected by knockdown of actin isoforms (F: n = 6; G and H: n = 5). Dendrite length (I) and number (J) are not altered by knockdown of individual actin isoforms (n = 5; sample size: I: shActα: 190, shActβ: 116, shActγ: 149, shCtrl: 136; J: shActα: 58, shActβ: 38, shActγ: 46, shCtrl: 49 cells). (K) Knockdown of Actα causes a reduction in soma size, whereas knockdown of Actβ and Actγ does not affect soma growth (***, P < 0.0001 for n = 5; sample size: shActα: 76, shActβ: 82, shActγ: 83, shCtrl: 69 cells). (L–N) Axon length is reduced after knockdown of Actα, Actβ, or Actγ (L: ***, P < 0.0001 for n = 5; sample size: shActα: 452, shCtrl: 380, untransduced: 328; M: ***, P < 0.0001 for n = 5; sample size: shActβ: 279, shCtrl: 227, untransduced: 219; N: ***, P < 0.0001 for n = 4; sample size: shActγ: 252, shCtrl: 228, untransduced: 180 cells). Statistical analysis in F–N was performed by one-way ANOVA with Dunn’s post-test. In B–H, data are shown as mean ± SEM. Bars: (A) 50 µm; (E) 10 µm.