Actβ-depleted motoneurons show smaller growth cones and reduced growth cone motility. (A) Motoneurons were transduced with shRNA expressing lentiviruses targeting actin isoforms and plated on laminin 221. Cells were fixed at DIV5 and stained with phalloidin and against Tau. Bars, 10 µm. (B and C) Growth cone area (B) and number of growth cone filopodia (C) are reduced after knockdown of Actβ (***, P < 0.0001 for n = 6; sample size: shActα: 96, shActβ: 54, shActγ: 67, shCtrl: 70 cells). (D) Time-lapse images representing dynamics of filopodia in growth cones of actin isoform–specific knockdown motoneurons (Videos 5–8). (E–H) Top panel shows maximum projection of 120 time-lapse images for measuring total changes in axonal growth cone movement in shCtrl (E), shActα (F), shActβ (G), and shActγ (H) transduced motoneurons, and lower panel shows multiple kymographs representing changes in filopodia/lamellipodia length (µm) over 10 min (time shown in seconds) along the red line inserted above. (I) Knockdown of Actβ reduces velocity of growth cone movements (***, P < 0.0001 for n = 3; sample size: shActα: 17, shActβ: 16, shActγ: 13, shCtrl: 15 cells). Statistical analysis was performed using one-way ANOVA with Dunn’s post-test. Bars: (D–H) 5 μm.