Labeling of newly incorporated barbed ends in axon shafts. (A) Protocol to label newly incorporated actin barbed ends. (B) Representative images of CAD cells after barbed end labeling. Note newly incorporated barbed ends at filopodial tips. (C) Top left: Microfluidic device to separate axons from somatodendritic compartments. Rhodamine (Rh)-actin was loaded only into the axonal chamber, and phalloidin staining was performed on both chambers as described in Materials and methods. Bottom: Rhodamine-actin and phalloidin labeling in somatodendritic (left) and axonal chambers (right). Note numerous small rhodamine-actin puncta in axons, indicating local incorporation of monomers (top right image). Also note some elongated structures (arrowheads in top right image) likely representing the rhodamine label incorporated into elongating actin filaments. (D and E) Kymographs from axons cotransfected with GFP:Utr-CH and mCherry:Mena or mCherry:Vasp and imaged near simultaneously. Note the colocalization of actin hotspots with Mena/Vasp (some marked with arrowheads). Colocalization is quantified in E. The plot depicts mean ± SEM. Bars: 10 µm (A and D); 50 µm (C). (F) Schematic showing actin trails elongating with their barbed ends facing the hotspots.