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. 2019 May 16;8:e46740. doi: 10.7554/eLife.46740

Figure 2. Misfolded PrP is detectable at the cell surface prior to lysosomal degradation.

(A) Cells expressing GFP-PrP and GFP-PrP* were incubated on ice with 200 nM extracellular Alexa647-conjugated Nb and Alexa647 fluorescence was measured by flow cytometry. Uninduced cells were analyzed in parallel and are shown in gray. (B) Diagram of experimental strategy to compare relative surface levels of GFP-PrP* and GFP-PrP. (C) Cells expressing GFP-PrP and GFP-PrP* were labeled with saturating amounts of anti-GFP Nb on ice, washed, and lysed under denaturing (SDS) or non-denaturing (CHAPS) conditions. Bound Nb was analyzed by immunoblotting relative to serial dilutions of purified recombinant Nb. (D) The indicated relative amounts of surface-derived complexes purified under non-denaturing conditions were analyzed by immunoblotting for GFP and FLAG. (E) GFP-PrP*-expressing cells treated for 96 hr with control or AP2-targeting siRNAs were surface-labeled with Alexa647-Nb and analyzed by flow cytometry. HEK293T cells without GFP-PrP* were analyzed for comparison (gray). (F) GFP-PrP*-expressing cells were transiently transfected with FusionRed-Dynamin S45N (Almeida-Souza et al., 2018) or empty vector for 24 hr prior to surface staining with Alexa647-Nb and analysis by flow cytometry. (G) GFP-PrP*-expressing cells were treated with 2.5 µg/ml Brefeldin A (BFA) for two hours prior to surface staining with Alexa647-Nb and analysis by flow cytometry.

Figure 2.

Figure 2—figure supplement 1. Characterization of Nb surface staining and isolation.

Figure 2—figure supplement 1.

(A) Cells expressing GFP-PrP were incubated on ice with varying concentrations of Alexa647-Nb, washed, and analyzed for Alexa647 fluorescence by flow cytometry. (B) GFP-PrP* on the cell surface was labeled on ice with Nb-FLAG, washed, and lysed under non-denaturing conditions. The lysate was then mixed with lysate from cells transfected with cytosolic GFP. The nanobody was subsequently immunoprecipitated (IP) via its FLAG epitope tag. The input, unbound and IP samples were analyzed by SDS-PAGE and immunoblotting using anti-GFP antibodies. No cytosolic GFP was recovered in the IP, illustrating that the Nb bound to surface GFP-PrP* does not exchange antigens after solubilization and during the IP. (C) GFP-PrP-expressing cells were incubated without or with extracellular trypsin to digest surface-localized proteins. The left panel shows the level of total GFP fluorescence analyzed by flow cytometry relative to uninduced cells. In the right panel, the trypsin-digested or undigested samples were surface labeled with fluorescent Nb and analyzed by flow cytometry. Note that surface GFP-PrP was quantitatively digested by trypsin as indicated by the fact that these cells show no detectable Nb fluorescence relative to cells that were not labeled with Nb. Thus, we can deduce that the remaining GFP fluorescence in the trypsin-digested cells seen in the left panel is due to the intracellular population of GFP-PrP. (D) Verification of AP2 knockdown after siRNA treatment, as part of the experiment in Figure 2E.