, 2007). However, the genetic deletion of Cofilin in the nervous system reduces neuronal cell proliferation and migration but not neurite formation ( Bellenchi et al., 2007). Moreover, the genetic ablation of ADF affects neither the development of the nervous system, nor the formation of neurites in

particular ( Bellenchi et al., 2007). Thus, so far, no actin filament modulator has been identified that regulates physiological neuritogenesis. Here, we observed that increased actin dynamics are correlated with and necessary for the emergence of neurites out of the neuronal sphere. Although required for neuritogenesis, microtubules mainly follow the lead of the progressively dynamic actin cytoskeleton. Genetic ablation of a single family of actin-regulating proteins, ADF and Cofilin (hereafter “AC PCI-32765 KO”), resulted in a failure of neuritogenesis due to profound cytoskeletal aberrations, including a blockade of F-actin retrograde flow and irregular microtubule growth. In the absence of AC proteins, pharmacological depolymerization of actin filaments enabled bundled microtubules to penetrate through the cell rim leading to neurite formation. The actin-severing

activity was primarily linked to actin retrograde flow and neurite formation. We conclude that AC regulates neuritogenesis http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html by driving actin turnover and organization, which is necessary for microtubule penetration and coalescence. We sought to characterize actin and microtubule dynamics during neurite formation. To date, such studies were hampered by the fact that fluorescently labeled actin could only be repetitively imaged in primary mammalian neurons

for short time periods or with low temporal resolution (Dent et al., 2007; Flynn et al., 2009). We therefore used neurons from Lifeact-GFP mice, which exhibited stable green fluorescent protein (GFP) fluorescence MG-132 solubility dmso to visualize actin dynamics with minimal photobleaching and phototoxicity (Riedl et al., 2010). To track polymerizing microtubules, we transfected Lifeact-GFP neurons with mCherry-tagged end-binding protein 3 (EB3-mCherry) (Akhmanova and Steinmetz, 2008). Within hours after plating, neurons assumed a characteristic “fried-egg” morphology (stage 1) (Dotti et al., 1988), with a circumferential actin-rich lamellipodium exhibiting moderate motility (Figure 1A, see Movie S1 available online). During neuritogenesis, filopodia became engorged with growing microtubules, expanded a growth cone, and progressed into a nascent neurite (Figure 1A). In addition, broad actin-based growth cone-like structures became more active and began advancing away from the soma, extending the membrane in their wake, which consolidated into a nascent neurite (Figure 1A). Initially, splayed microtubules closely trailed advancing actin structures and later coalesced into bundles as the neurite took shape (Figures 1A and S1A, Movie S1).