Figure 2.

APPL trafficking and processing in cultured Manduca neurons corresponds to their stage of outgrowth. Neurons harvested from the CNS of fifth instar larvae were grown as dispersed cultures on glass coverslips for 1–14 days in vitro, then fixed and immunolabeled with a combination of anti-nAPPL (green) and anti-cAPPL (magenta) antibodies. Anti-nAPPL and anti-cAPPL are shown individually as gray scale images but are shown in green and magenta (respectively) in the merged images, whereby co-immunolabeling appears white (right hand column). (A) By Day 1, neurons had begun to extend numerous processes with exploratory growth cones (arrows); full-length APPL (white immunolabeling) accumulated in the leading filopodia, while C-terminal fragments (magenta arrowheads) were relatively more abundant throughout the growing neurites and N-terminal fragments (green arrowheads) were enriched in the cell bodies. APPL holoprotein also accumulated in a population of large perinuclear cytoplasmic vesicles (white arrowhead). (B) Higher magnification view of the neuron shown in (A); numerous small vesicles containing only N-terminal fragments (green arrowheads) or C-terminal fragments (magenta arrowhead) were interspersed among the larger vesicles containing the holoprotein (white arrowheads). (C) By day 3, many neurons had extended primary neurites with enlarged growth cones; as is apparent in the merged image, APPL holoprotein (white immunolabeling) continued to be enriched at the leading edges of the growth cones and in some filopodia (arrows), while numerous smaller vesicles containing either N-terminal or C-terminal fragments were distributed throughout the neuronal somata and processes. (D) By day 8, most neurons were no longer undergoing active outgrowth. Full-length APPL was still abundant within vesicles in their somata (white immunolabeling in the merged image) but was no longer concentrated in the distal tips of their processes (open arrows). By comparison, vesicles containing C-terminal fragments (magenta arrowheads) were diffusely distributed throughout the neurons and their processes, while N-terminal fragments (green arrowheads) remained relatively more abundant in the somata. (E) By 12 days, most neurons were either undergoing retraction (open arrows) or initiating degeneration (not shown). APPL holoprotein was almost completely absent from their distal processes, as were vesicles containing N-terminal fragments, whereas vesicles containing C-terminal fragments (magenta) could still be detected throughout the neurons. These results suggest that full-length APPL is selectively transported to the distal tips of developing neurons during periods of active outgrowth. Scale bar = 2 μm in (B); 10 μm in all other panels.