The protrusion of microtubules is essential for neurite formation as low levels of the microtubule-destabilizing drug nocodazole attenuated the neurite-restoring effects of cytochalasin D and latrunculin see more B ( Figure S5). Instead, manipulations that were targeted either to stimulate

integrin signaling or to bundle actin filaments, which restore neurite formation in Mena/VASP/EVL KO neurons ( Dent et al., 2007), did not enable neurite formation in AC KO neurons ( Figure S6). We conclude that the drastic F-actin disorganization in AC KO neurons obstructs intracellular space and misdirects microtubule growth patterns. Furthermore, a pharmacological depolymerizing activity bypasses the need for AC proteins, allowing microtubules to coalesce and to radially protrude to generate neurites. Although in some instances the function of ADF and Cofilin are overlapping (Hotulainen et al., 2005), ADF depolymerizes actin filaments better than Cofilin, whereas Cofilin severs filaments better than ADF (Bernstein and Bamburg, 2010). Therefore, we determined the individual contributions of ADF and Cofilin to neuritogenesis. First, we examined the development of neurons with either monoallele ADF

selleck compound expression (NesCre+/−, ADF+/−, cofilinflox/flox) or monoallele cofilin expression (NesCre+/−, ADF−/−, cofilinflox/+). ADF monoallele expression resulted in defective neuritogenesis with a significant increase in the percentage of cells in stage 1 ( Figures S7A and S7B). In contrast, cofilin monoallele expression conferred wild-type-like neuronal development, with the majority of neurons in stage 2 or stage 3 ( Figures S7C and S7D). Consistently, reintroduction

of ADF into AC KO neurons only partially restored neurite already formation in AC KO neurons, whereas Cofilin reintroduction almost completely reversed the neuritogenesis defect, resulting in cells with wild-type morphology in cell culture ( Figures 7A and 7B). Moreover, Cofilin re-expression restored normal neuronal development in AC KO cortical slices ( Figures 7C, 7D, and S7E). Analysis of the F-actin organization revealed that both ADF and Cofilin restored the gross organization of actin architecture. The percentage of cells extending filopodia increased 2-fold in ADF or Cofilin-transfected AC KO neurons ( Figures 7E and 7F). However, kymograph analysis of live-cell imaging of AC KO neurons cotransfected with Lifeact-GFP revealed that only Cofilin expression increased actin retrograde flow to 4.0 ± 1.0 μm/min, nearly a complete rescue, while ADF only partially increased actin retrograde flow to 2.9 ± 1.2 μm/min, a 65% rescue ( Figures 7E and 7G). Thus, while ADF and Cofilin are equally adept at stimulating filopodia formation, Cofilin has a higher propensity for driving actin retrograde flow and neuritogenesis.