Dynamic Organization of Endocytic Pathways in Axons of Cultured Sympathetic Neurons

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Abstract
Despite the wealth of information about endocytic pathways in non-neuronal cells, little is known about these crucial sorting, recycling, and degradative pathways in neurons. In this report, we analyzed in detail the dynamic steady-state organization of endocytically derived organelles as they progress through the endosomal–lysosomal pathway in axons of live cultured sympathetic neurons. By ratiometric imaging of neurons endocytically labeled with the pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), we demonstrate a trimodal frequency distribution of endocytic organelle pH in axon shafts, indicating two rapid acidification steps in the progression from endocytosis to the lysosome. Axonal branch points display a unimodal organelle pH distribution (mean 6.4), implicating them as meeting places for endocytic organelles and Golgi-derived vesicles or as sorting sites. By following endocytic organelle traffic retrogradely from growth cone to soma, we identified significant transition points in the pathway. Growth cones exhibit a unimodal pH distribution comprised mainly of acidified recycling/sorting endosomes (mean 6.3). However, organelles in the axon shaft immediately adjacent to the growth cone display the distinct trimodal pH distribution of the axon, suggesting that important sorting events occur between these domains. An abrupt increase in organelle acidification occurs in the distal axon 50–150 μm from the growth cone, demonstrating a discontinuous spatial gradient of acidification along axons. Immunofluorescence microscopy reveals that the lysosomal glycoprotein LEP100 is present in axons and is concentrated in two important regions: the proximal axon where the endocytic organelle population is largely acidified, and the same region of the distal axon where substantial acidification occurs.