Figure 5

Transferrin-positive endosome abnormalities and reduced recycling of the TrkA receptor in PC12-U18666A cells. A. PC12 cells were transfected with a Rab5-EGFP plasmid (which labels early endosomes in green) and after 24 hrs were treated (U18666A) or not treated (Control) with U18666A (2 μg/ml) for another 24 hrs. To load the cells with transferrin (labeling recycling endosomes in red), PC12 cells were serum-starved for 1 hr and treated with transferrin Alexa Fluor 555 (2 μg/ml) for 2 hrs at 37°C. Cells were fixed with paraformaldehyde and visualized with a confocal microscope. Scale bar, 5 μm. B. The fluorescence from Rab5-EGFP (n = 16-24 cells, from two different experiments) and transferrin (n = 57-60 cells, from two different experiments) associated with the perinuclear region of PC12 cells was measured and plotted. C, control PC12 cells. U18, PC12 cells treated for 24 hrs with U18666A (2 μg/ml), *p < 0.0001. C. Immunoendocytosis (2 hrs at 37°C) of endogenous TrkA (TrkA endogenous) and TrkA-Flag in PC12 cells not treated (Control) or treated (U18666A) for two days with the drug (2 μg/ml). The arrows indicate the presence of larger endosomes in U18666A-treated PC12 cells than in non-treated cells. D. Volume quantification of TrkA-positive endosomes of PC12 cells non-treated (C) or treated (U18) with U18666A (2 μg/ml). For endogenous TrkA-labeled endosomes (TrkA endo), a total of 63-69 vesicles were considered (from 6-7 representative cells from two different experiments). *p < 0.0001. For TrkA-Flag-labeled endosomes, a total of 15-19 endosomes were considered (from 2 representative cells). *p < 0.0003. E. Immunoendocytosis of TrkA-Flag was allowed to proceed for 2 hrs at 37°C in the presence of NGF (2 hrs inter). The Alexa Fluor 488-conjugated anti-Flag monoclonal antibody was then washed from PC12 cells by treatment with cooled EDTA. PC12 cells were fixed and mounted. For recycling experiments, immunoendocytosis of TrkA-Flag was allowed to proceed for 60 min, and the Alexa Fluor 488-conjugated anti-Flag monoclonal antibody was then washed from PC12 cells by treatment with cooled EDTA and further incubated with an anti-Alexa Fluor 488 antibody for 60 min at 37°C (60 min recycling), fixed and mounted. F. The fluorescence associated with PC12 cells after the treatments indicated in E was quantified and plotted. 2 hrs inter, 2 hrs internalization. 60 min rec, 60 min recycling. To measure the fluorescence associated with PC12 cells after 2 hrs of internalization of the Alexa Fluor 488-conjugated anti-Flag monoclonal antibody, 61-67 cells were considered from two different experiments. To measure the fluorescence associated with PC12 cells after 60 min recycling, 23-45 cells were considered from two different experiments. *p < 0.0023. G. To visualize the co-localization of TrkA-Flag with transferrin, PC12 cells not treated (control) or treated (U18666A) with the drug were incubated with the Alexa Fluor 488-conjugated anti-Flag monoclonal antibody and Alexa Fluor 555-conjugated transferrin (60 μg/ml) for 2 hrs in the presence of NGF and fixed. H. To visualize the co-localization of TrkA-Flag with LAMP2, PC12 cells not treated (control) or treated (U18666A) with U18666A were incubated with the Alexa Fluor 488-conjugated anti-Flag monoclonal antibody for 2 hrs in the presence of NGF, fixed and immunostained with a polyclonal antibody against LAMP2 (lysosomal marker).