Figure 5. Retrogradely transported TrkA⁺ endosomes within cell bodies evolve from MVBs into simple, single-membrane vesicle structures.

(a) Schematic of the pulse-block assay. The Flag-TrkA assay is performed in Ntrk1^Flag sympathetic neurons cultured in compartmentalized microfluidic chambers as in Figure 1—figure supplement 1A using pre-conjugated anti-Flag antibody and Protein A-5 nm gold. Nocodazole is applied to distal axons 25 min post-NGF application to block retrograde transport. Neurons are fixed at indicated time points and processed for EM. (b) The Flag-TrkA assay was performed in WT neurons, Ntrk1^Flag neurons treated with either DMSO or nocodazole in distal axons (25 min post NGF). Cells were fixed at indicated time points and processed for EM. The number of gold particles per section per cell was quantified (n = 3, Results were pooled from over 150 Flag-gold particles). (c) The pulse-block assay was performed as in (a), and membrane topology of the Flag epitope was assessed. A schematic of the inner- or outer-leaflet position for membrane of ILVs, the limiting membrane and SVs is shown on the right. (d,e) The pulse-block assay was performed and the distribution of retrogradely transported Flag-TrkA gold particles in MVBs, single-membrane vesicle structures (SVs) and lysosomes within the cell body over time was assessed (e) Results were pooled from over 150 Flag-gold particles from four samples for each time point. Representative images of retrograde Flag-TrkA in each of the three membrane compartments are shown in (d). (f) Sympathetic neurons in compartmentalized cultures were incubated with transferrin-gold (6 nm) in distal axons and the pulse-block assay was performed. The distribution of retrograde transferrin-gold in MVBs, SVs and lysosomes was assessed by EM. For (d–f), over 150 endosomes were counted for each condition at each time point in four independent experiments. Scale bar: 100 nm. Data are represented as mean ± SEM. ***p<0.001 by two-way ANOVA with a Tukey’s post-hoc test (e) or a two-tailed unpaired Student’s t test (c). See also Figure 5—figure supplement 1.

Figure 5—figure supplement 1. Nocodazole treatment effectively blocks microtubule-dependent axonal trafficking and retrograde Flag-TrkA transport.

(a) Quantification of retrograde Flag-TrkA localization in MVB, SV and lysosome in cell bodies over time (n = 4,>200 gold particles scored for each time point). Data for the 1 hr time point is the same as in Figure 1C. (b) The Flag-TrkA assay was performed in compartmentalized sympathetic neurons with DMSO or nocodazole (10 μM) applied at the time of NGF application in DA. Accumulation of Flag-TrkA punctae in cell bodies and distal axons was assessed 3 hr post-NGF application (n = 3). Compared to the vehicle control, very few, if any, Flag punctae were observed in nocodazole treated cells. Scale bar: 20 μm. (c) The Flag-TrkA assay was performed in compartmentalized sympathetic neurons with nocodazole (10 μM) applied 25 min post-NGF application in DA. Movement of retrograde Flag-TrkA endosomes was monitored by live imaging in the middle grooves of microfluidic chambers (n = 3). A representative kymograph is shown (left panel). The rate of movement of individual Flag-TrkA endosomes in distal axons at the time of nocodazole application and 20 min afterwards was measured (Right panel). Retrograde movement halted ~15 min post-nocodazole application. Scale bar: 20 μm; 5 min. (d,e) Alexa-488-labeled CTB was applied to the cell body compartment of compartmentalized neuronal culture in the presence of nocodazole either in the cell body or distal axon compartment. The endocytic trafficking of CTB-Alexa488 to lysosomes (c) and axonal movement of CTB (d) were assessed. Scale bar: 10 μm (c). 10 μm; 1 min (d). **p<0.01 using a two-tailed paired Student’s t test.