(A) Compartmentalized sympathetic neurons were deprived of NGF for 18h NGF, then distal axons were incubated with NGF (100 ng/ml), NT3 (1000 ng/ml), or no neurotrophin. Surviving cells were counted 36-48h later. Shown are means +/− SEM, (n=3).
(B) Mass cultures of sympathetic neurons were NGF-deprived overnight and then stimulated with NGF or NT-3 for 20 min. Lysates were subjected to SDS-PAGE and immunoblotted for the indicated proteins. Densitometric analysis indicates that canonical TrkA effectors are similarly activated by NGF and NT3. Upper and lower blots represent different samples. Shown are means +/− SEM, (n=3),.
(C) The Flag-TrkA endosome transport assay was done in compartmentalized sympathetic neurons; distal axons were incubated with NGF or NT3 for 3h. The percentage of cell bodies (CB) containing Flag-TrkA punctae was quantified and data are represented as mean +/− SEM, (n=4). Scale bar represents 10 μm.
(D) The Flag-TrkA endosome transport assay was performed to assess the extent of internalization of Flag-TrkA and formation of Flag-TrkA endosomes following a 30 min treatment with NGF or NT3 on distal axons of compartmentalized sympathetic neurons. The number of internalized Flag-TrkA endosomes was counted per 10 μm of axon for each condition. Scale bar represents 10μm. Statistical analysis was done using one-way ANOVA followed by Tukey's post-hoc test. ** p < 0.01, *** p < 0.0001.

(A) Early endosomes (top panels) were purified from cultured rat sympathetic neurons deprived of NGF for 18h then treated for 20 min with NGF (100 ng/ml) or NT3 (1000 ng/ml) or left untreated. Levels of total cofilin, Ser³-phosphorylated cofilin, and Rac1 were assessed by immunoblot analysis. Relative levels of cofilin and inactive cofilin (Ser3 phosphorylated) associated with NGF and NT3 endosomes were quantified. Whole cell lysates (bottom panels) from sympathetic neurons treated with NGF and NT3 were analyzed for cofilin and phosphor-cofilin. Data are represented as mean +/− SEM, (n=3), ** p< 0.01, *** p<0.0001.
(B) Colocalization of internalized Flag-TrkA endosomes (red) and cofilin (green) was assessed by immunocytochemistry of distal axons of sympathetic neurons that were untreated, or treated with either NGF or NT3. Data are represented as mean +/− SEM, (n=5), ** p< 0.01. Scale bar represents 10μm.
(C) Lentivirus infected sympathetic neurons were grown in microfluidic chambers, and the retrograde transport of Flag-TrkA endosomes was assessed. Neurons were counterstained with FITC-phalloidin. Scale bar represents 10μm.
(D) The effect of cofilin knockdown on NGF-dependent retrograde survival of neurons was tested for sympathetic neurons grown in microfluidic chambers. Neurons were allowed to project axons into distal chambers and then were infected with either a control lentivirus or a virus expressing an shRNA against cofilin. Survival was monitored 36h to 48h later under conditions in which NGF was exposed to both cell body and distal axon compartments, or NGF was exposed exclusively to the distal axon compartment. Data are represented as mean +/− SEM, (n=4), ** p< 0.01.
(E) Inhibition of actin polymerization by LatA rescues the TrkA transport defect of cofilin knockdown neurons. Neurons were treated as in (D) with the addition of a 30 min pretreatment of vehicle or LatA in distal axons prior to performing the Flag-TrkA retrograde transport assay. Data are represented as mean +/− SEM, (n=3). Scale bar represents 10μm. Statistical analysis was done using Student's t test with the exception of (C), which was done using one-way ANOVA followed by Tukey's post-hoc test. * p< 0.05.

(A) Compartmentalized sympathetic neurons were infected with control or RacN17 virus, NGF-deprived overnight, and subjected to the Flag-TrkA transport assay in the absence or presence of NGF for 3h. Confocal images of cell bodies are shown. Scale bar represents 10μm.
(B) Percentage of cell bodies containing Flag-TrkA punctae following distal axon incubation with vehicle or NGF. Shown are means +/− SEM, (n=3), *** p<0.0001.
(C) Sympathetic neurons were grown as above and infected with a control or RacN17 virus. Neuronal survival was measured and data are represented as mean +/− SEM. (n=3), ** p<0.01.
(D) Compartmentalized sympathetic neurons were infected with a control virus or a virus encoding a constitutively active form of Rac1 (RacV12). Neurons were NGF deprived overnight, subjected to the Flag-TrkA transport assay, and stimulated on distal axons for 3h with vehicle, NGF, or NT3. Confocal images of cell bodies are shown. Scale bar represents 10μm.
(E) Percentage of cell bodies containing Flag-TrkA punctae following distal axon stimulation with vehicle, NGF or NT3. Data are represented as mean +/− SEM, (n=3), *** p<0.0001, **p<0.01.
(F) Compartmentalized neurons were infected with control or RacV12 virus and were treated as indicated, and then neuronal survival was measured. Data are represented as mean +/− SEM, (n=4), *** p<0.0001, ** p<0.01, * p<0.05. Statistical analysis was done using one-way ANOVA followed by Tukey's post-hoc test.

(A) PC12 cells were stimulated with NGF or NT3 for 10 min in DMEM buffered at pH 7.0, pH 6.0 or pH 5.5. Cells were subsequently lysed and subjected to immunoblotting for TrkA phosphorylated on tyrosine 490 (P-TrkA). Densitometric analysis was performed and P-TrkA levels were normalized against the amount of actin. Data are represented as mean +/− SEM, (n=3), ** p<0.01, * p<0.05. Statistical analysis was done using one-way ANOVA followed by Tukey's post-hoc test.
(B) Radioligand binding assays were performed in which COS7 cells in 24 well plates, either untransfected or transfected with full length TrkA, were incubated with 1 nM ¹²⁵I-NGF or ¹²⁵I-NT3 in media buffered to the pH values indicated. Data are represented as mean +/− SEM, (n=3). Similar results were obtained in experiments using 0.2 nM ¹²⁵I-NGF and ¹²⁵I-NT3 (not shown).
(C) Compartmentalized sympathetic neurons were NGF-deprived overnight, and the Flag-TrkA transport assay was performed. Distal axons were stimulated with NGF or NT3 alone or with NGF or NT3 in the presence of NH₄Cl or bafilomycin for 3h. Similar experiments were done using neurons that were infected with either a control or RacN17 virus. Scale bar represents 10μm.
(D) Quantification of (C).
(E) Colocalization of internalized Flag-TrkA endosomes (red) and cofilin (green) was assessed by immunocytochemistry of distal axons of sympathetic neurons treated for 20 minutes with NT3 in the presence of either DMSO (vehicle control) or bafilomycin. The number of cofilin positive Flag-TrkA endosomes in distal axons was quantified. Data are represented as mean +/−SEM, (n=5), ** p< 0.01. Scale bar represents 10μm. Statistical analysis was done using Student's t test.
(F) Distal axons of compartmentalized sympathetic neurons were stimulated with NGF or NT3 in the presence or absence of bafilomycin for 3h. Neurons were subsequently fixed and subjected to the GST-PAK immunoassay to visualize Rac1-GTP punctae. Quantification represents number of Rac1-GTP punctae per cell body or per 30 μm of axon. Scale bar represents 10μm.

(A) Protein from purified PC12 TrkA endosomes was analyzed by immunoblot.
(B) Sympathetic neurons were grown in microfluidic chambers and pretreated for 30 min with Latrunculin A (LatA) (10μM) applied to the distal axon (DA) chamber. Retrograde transport of TrkA endosomes to cell bodies was assessed using the Flag-TrkA endosome transport assay. Transported Flag-TrkA endosomes are shown in red and neurons were counterstained with FITC-phalloidin. Scale bar represents 10μm.
(C) Sympathetic neurons grown in microfluidic chambers were treated with Jasplakinolide (10μM) for 30 min prior to assessment of Flag-TrkA retrograde transport. Internalized and transported Flag-TrkA endosomes are shown in red; neurons were counterstained with NF200. Retrograde signaling endosome transport to the cell body and internalized TrkA in distal axons is quantified (upper and lower right, respectively). Scale bar represents 10μm. Statistical analysis was done using the Student's t test **p<0.01.
(D) TrkA endosomes are associated with an F-actin depolymerization activity. Control beads or bead-bound Flag-TrkA endosomes were incubated with 2μM F-actin. After 15 min, beads were removed, F-actin and G-actin were separated by ultracentrifugation, and protein was analyzed by SDS-PAGE and Coomassie staining.

(A) Dissociated sympathetic neurons were stimulated with NGF (100 ng/ml) or NT3 (1000 ng/ml) for 20 min. Cell lysates were prepared and subjected to GST-PAK pull down and were immunoblotted for Rac1. Densitometry was performed and signal intensities were normalized against total Rac1. Data are represented as mean +/− SEM, (n=3). Statistical analysis was done using Student's t test. ** p< 0.01.
(B) Compartmentalized sympathetic neurons were subjected to the Flag-TrkA endosome assay and distal axons were incubated with vehicle, NGF or NT3 for 3h and then processed with the GST-PAK immunoassay to simultaneously visualize Flag-TrkA (red) and Rac1-GTP (green). Confocal images of distal axons (DA), middle axons (MA) and cell bodies (CB) are shown. Scale bar represents 10μm. Quantification of total and colocalized signal is shown below representative pictures.

Sympathetic neurons were grown in microfluidic chambers and infected with a RacN17 virus or a control virus. Two days later, neurons were subjected to the Flag-TrkA labeling assay in the presence of NGF (100 ng/ml). The effect of Rac1N17 on cofilin localization was examined by immunocytochemistry; internalized Flag-TrkA endosomes are labeled red and cofilin is labeled green. The percentage of cofilin positive Flag-TrkA endosomes was quantified. Shown are means +/− SEM, (n=5). Scale bar represents 10μm. Statistical analysis was done using Student's t test. ** p< 0.01