Endocytic tubules and vacuoles in the growth cone central domain. (A) Time-lapse confocal images of FM 5-95 internalization show dye incorporation into elongated tubules (green arrows, 0:02) and vacuoles (yellow arrows, 0:01 to 0:03) that correspond to the timing of surface membrane labeling. Tubules (green arrowheads, 0:06 to 0:08) and vacuoles are most often stationary but occasionally become motile and contact one another (yellow asterisk, 0:10 to 0:12). See Additional file 13 for the corresponding time-lapse movie. (B) Confocal images show FM 5-95 labeled vacuoles associated with dorsal (red arrow) and ventral (yellow arrow) surface membranes. The boxed region on the left is magnified at the right, which shows two different confocal z-sections of the same growth cone. Dye-labeled vacuoles are either seen in the dorsal (upper magnified panel) or ventral (lower magnified panel) confocal sections of the growth cone. The numerous vesicles in the upper-right lamellipodium of the far left panel originated from small vesicle hot-spots. Scale bars, 5 μm (left), 1 μm (right). (C) Time-lapse confocal images show that FM dye-labeled vacuoles are continuous with the plasma membrane for extended time periods. The time (minutes:seconds) following the initial dye pulse is indicated in each frame. In this modified dual-labeling assay, a first micropipette delivered a focal pulse of FM 2-10 at 0:00 (green, left panel), which rapidly labels several small vesicles and a vacuole (0:04, white arrow). A subsequent pulse of FM 5-95 (red), applied from a second micropipette at 0:12, incorporates into the same vacuole (0:14, white arrow). Scale bar, 5 μm.