Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 4
An intact Golgi is not essential for the delivery of Mut-PrP to LysoTracker positive vesicles. (a) Golgi disruption by Brefeldin A (BFA) (5 μg/mL for 3 h) was confirmed by the absence of Wt-PrP signal on the plasmalemma of HeLa cells expressing Wt-PrP::GFP, compared with vehicle-treated cells sampled at the same time point. (b) Functional disruption of Golgi by BFA was biochemically confirmed by the absence of complex glycans (*) on Wt-PrP, following Western blot of total cell lysates, compared with vehicle-treated (−) cells. Mut-PrP does not acquire complex sugars and as such is unaffected by BFA. PrP was detected with D13 antibody. (c) Experimental paradigm; times are relative to the completion of the transfection protocol. BFA (5 μg) was added at 5.5 hours, at the earliest detection of PrP signal, LysoTracker Red (50 nM) was added 30 minutes prior to the start of imaging at 9 hours, and images were collected by 10 hours. (d) Immunofluorescence of live cells expressing GFP-tagged Mut-PrP after treatment with vehicle or BFA. Green (PrP::GFP) and red (LysoTracker) channels are displayed as merged. Separate channels for the boxed areas of BFA treated cells are magnified and displayed to the right of the merged images. Correlation coefficient analysis revealed a statistically significant increase in colocalization of PrP and LysoTracker after BFA treatment for both Wt-PrP and Mut-PrP. Wt-PrP veh = , BFA = ( cells, ); Mut-PrP veh = , BFA = ( cells, ). (e) BFA treatment increases the insoluble fraction of PrP. N2a cells stably expressing 3F4-tagged Wt- or Mut PrP were treated with BFA (5 ug/mL for 3 h) or vehicle, and the insoluble fraction of PrP was separated by centrifugation at 16,000 ×g, and equal volumes of the total (T), supernatant (S), and pellet (P) fractions were loaded and subjected to Western blot using the 3F4 mAb. (f) Densitometric quantification of (e), using Image One (BioRad) software. The fraction of insoluble Wt-PrP was significantly increased after BFA treatment (, ), while the increase in insoluble Mut-PrP did not reach significance ().
