PrPc and CD59 occupy different microdomains in the plasma membrane, and PrPc cycles rapidly from the plasma membrane to the internal membranes. (Ai) Fixed and permeabilized day-7 erythroblasts were dual stained with PrP mAb (red) after use of conversion antibody 1/10 and CD59 (green) and subsequently stained with FITC and TRITC secondary antibodies. Overlays were prepared and highlighted areas from merge magnified to show (Aii) internal PrP-containing vesicles (arrows) and (Aiii) the plasma membrane. The profile (Aiv) of PrP and CD59 fluorescence colocalization in the surface of the cell membrane was analyzed by quantitation software (Leica). Scale bars equal 10 μm (Ai), 5 μm (Aii,iii). (B) Internalization assays were performed to compare the internalization of PrP and CD59 in erythroid cells. After allowing binding of the relevant antibody to the cell surface, cells were incubated at 37°C for up to 60 minutes to allow endocytosis to occur. (Bi) PrP is internalized within 10 minutes endocytosis (grayscale; arrow). (Bii) CD59 is not internalized after 60 minutes endocytosis (grayscale). (Biii) Dual staining of PrP (green) and TfR (red) after 10 minutes of endocytosis (arrow indicates internal colocalization). (Biv) Internalized CD63 was observed inside the erythroblasts at 5 and 10 minutes. Scale bars equal 10 μm. (C) PrP mAb (red) was subject to the internalization protocol, fixed and permeabilized, incubated with conversion antibody (1/10), and then dual stained with CD63 mAb (green). Cell-surface and internalized PrP only is shown stained in red and total CD63 in green. Areas of colocalization are yellow. (Ci) Internalized PrP after 5 minutes (red) and CD63 (green) colocalize in a vesicle-like structure just under the plasma membrane (arrow). (Cii) A region of interest (ROI) was drawn through the cell to include internalized PrP, and a profile of green and red fluorescence along this ROI was constructed using Leica quantiation software. (Ciii) Internalized PrP after 20 minutes (red) and CD63 (green) colocalize deep within the cell. Grayscale image shows areas of colocalization between PrP and CD63 only (arrows). (Civ) An ROI was drawn through a cell to include internalized PrP, and a profile of green and red fluorescence along this ROI was constructed using Leica quantiation software. Scale bars equal 10 μm.
