Vacuole and tubule unloading or recycling back to the plasma membrane. (A) Time-lapse confocal images of FM 5-95 internalization show the disappearance of a vacuole in the growth cone central domain. Focally applied FM 5-95 (applied at 0:00) immediately incorporates into several vacuoles (0:02). In subsequent frames, one vacuole narrows (yellow arrows, 0:04) and later disappears (0:06 to 0:08). Note that the size and location of other nearby vacuoles remains unchanged (red arrowheads). (B) Time-lapse confocal images of fluorescent dextran internalization show sudden dye unloading by an elongated tubule in the growth cone periphery (yellow static arrows). The dextran pulse was applied at 0:00 and washed away from 0:10 to 0:15. Note that the labeled tubule disappears within 40 to 50 s of forming (0:51, yellow asterisk). (C) Time-lapse confocal images show rapid unloading of dextran-containing vesicles. The focal pulse was applied and washed away as in (B). Within 20 to 30 s of vesicle formation, nascent vesicles (yellow, red, blue static arrowheads) begin to unload dye. For all examples (A-C), the boxed regions in the left panels are shown at higher magnification on the right and the time after the focal dye pulse (minutes:seconds) is indicated in each frame. Time-lapse movies for (A-C) are shown in Additional file 14. Scale bars, 5 μm (left), 1 μm (right).