Fig. 3. Bulk endocytosis in the growth cone is linked to membrane ruffling and depends on actin dynamics, PI3-kinase activity and cholesterol levels. (A) Growth cones (2 DIV) that were either left untreated (KRH) or subjected to various treatments before incubation for 1 minute with KRH in the presence of FM4-64 are shown. Incubation of growth cones at 4°C during FM4-64 application prevents dye uptake (KRH 4°C). Incubation with either 2 mM EGTA-containing solution devoid of Ca²⁺ for 30 minutes (EGTA) or BFA (10 µg/ml) for 1 hour does not affect FM4-64 internalization. Treatments with CytD (10 µM, 15 minutes), LY294002 (50 µm, 30 minutes) or MβCD (5 mM, 3 minutes) inhibit FM4-64 uptake. (B) Time-lapse DIC imaging of a growth cone (2 DIV) incubated for 130 seconds in KRH prior to exposure for 1 minute to KRH containing FM4-64. The last panel on the right represents the overlay of the DIC image taken at 190 seconds with the FM4-64 staining (red). The dye is internalized in the region of the growth cone that is associated with active plasma-membrane ruffling (arrowhead). (C) Quantification of FM4-64 uptake in growth cones that were treated as shown in A. The amounts of FM4-64 loaded in 2-DIV growth cones during a 1-minute incubation in high K⁺ (KCl) or remaining after incubation in KRH for 30 minutes at 37°C (unloading) are also reported (mean ± s.e.m.; n=30-40 growth cones per treatment; *P<0.001, Dunnett's test for untreated growth cones vs growth cones subjected to various treatments). (D,E) Growth cones loaded with FM4-64 (KRH, 1 minute; red) and retrospectively stained with either FITC-phalloidin to visualize F-actin (D; green) or filipin to visualize cholesterol (E; blue). The overlay between the corresponding fluorescence and DIC images is shown in the right panels. The arrowhead in D points to a ruffling focus colocalizing with FM4-64 labeling. Scale bar: 10 µm.