Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 3
Time-lapse photomicroscopy of PrP::GFP trafficking to lysosomes. (a) At COS-7 cells were transfected with Mut-PrP::GFP over 3 h, the media were replaced, and imaging started. (a) displays frames of live video collected from min (5 h) to 420 min. Images were extracted as individual frames from supplement Video S1, using ImageJ. (b) Mut-PrP::GFP, as in (a), but in the presence of 30 nM LysoTracker Red. Frames were collected between (7 h) and 560 min. Arrows indicate the first appearance of yellow puncta, signifying Mut-PrP delivery to LysoTracker Red positive vesicles. Images were extracted from supplement Video S2. Magnified insets, approximately 2x original image. (c) Wt-PrP::GFP in the presence of LysoTracker Red imaged as in (b) for Mut-PrP, but with a time window of 400 to 950 min, to capture PrP entry into lysosomes. Wt-PrP::GFP is detected on the plasmalemma (arrowheads) prior to its colocalization with LysoTracker Red positive vesicles (arrows), at a much delayed time point than Mut-PrP (750 versus 460 min). Images extracted from supplement Video S3. Magnified insets 4x original image, to compare with (b).
