Endocytic vesicle formation at self-membrane contacts requires F-actin. (A) Summary of axon growth rates in vehicle treated controls and after treatment with cytochalasin D during a one-hour growth assay. Data are the mean ± standard error of the mean and the number of axons measured is indicated for each condition. N.S. (no significant difference), P > 0.05, *P < 0.0001, One-way ANOVA, Tukey's post-test. (B) Representative confocal images of control (DMSO) and cytochalasin D (CytoD, 30 nM) treated growth cones show the distribution of F-actin and microtubules, as detected by Alexa555-phalloidin (red) and anti-β-tubulin immunolabeling (green). Note the fewer peripheral processes and reduced F-actin after CytoD. Scale bar, 5 μm. (C) Time-lapse confocal images after a FM 5-95 dye pulse show a motile filopodium (yellow arrows) contacting and fusing with the growth cone peripheral plasma membrane. The self-membrane contact fails to trigger vesicle formation. The boxed region is magnified in the right panels and the time (minutes:seconds) after the initial membrane labeling is indicated in each frame. The corresponding time-lapse movie is shown in Additional file 10. Scale bars, 5 μm. (D-E) Quantitative analysis of membrane retrieval at self-membrane contact sites (filopodial - lamellipodial and filopodial - filopodial contacts) in untreated and cytochalasin D-treated (30 nM) growth cones. The percentage of total self-membrane contact sites associated with membrane retrieval is shown in (D), and the frequency of membrane retrieval events (per minute) occurring at self-membrane contact sites is shown in (E). (F) Frequency (per minute) measurements for the total number of self-membrane contact events, including those not associated with membrane retrieval, measured during the focal membrane labeling assays. For (D-F), data are the mean ± standard error of the mean and the number of growth cones is indicated for each bar. N.S., P > 0.05, **P < 0.01, t-test.