Normal prion protein trafficking in cultured human erythroblasts by Rebecca E. Griffiths, Kate J. Heesom, and David J. Anstee Blood Volume 110(13):4518-4525 December 15, 2007 ©2007 by American Society of Hematology

PrPc expression in human erythroid cells cultured in vitro. PrPc expression in human erythroid cells cultured in vitro. (A) Erythroblasts were harvested on days 6, 8, and 11 of culture. After seeding overnight onto poly-L-lysine–coated slips, cells were fixed and permeabilized and then incubated with PrP mAb ICSM18 and subsequently with goat anti-mouse FITC. Scale bars equal 10 μm. (B) Day-6 erythroblasts were fixed and permeabilized. Dual staining of cells with PrP mAb ICSM18 show in green, and ER marker calnexin (i), Golgi marker giantin (ii), and lysosomal marker LAMP-1 (iii) shown in red. Bottom row (iv) shows PrP mAb (red) dual stained with CD63 mAb (green). Colocalization is highlighted in the grayscale image. Scale bars equal 10 μm. Rebecca E. Griffiths et al. Blood 2007;110:4518-4525 ©2007 by American Society of Hematology

PrPc and CD59 occupy different microdomains in the plasma membrane, and PrPc cycles rapidly from the plasma membrane to the internal membranes. 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. Rebecca E. Griffiths et al. Blood 2007;110:4518-4525 ©2007 by American Society of Hematology

PrPc colocalizes with tetraspanins CD81 and CD82 at the plasma membrane. PrPc colocalizes with tetraspanins CD81 and CD82 at the plasma membrane. (Ai) Fixed and permeabilized day-6 erythroblasts were dual stained with PrPc mAb (red) and CD81 mAb (green). Overlays were prepared and the highlighted area magnified to show (Aii) an internal colocalized vesicle (arrow) and the plasma membrane. (Aiii) ROIs were drawn through a cell (ROI1) and along the cell surface (ROI2), and profiles of green and red fluorescence along these ROIs were constructed using Leica quantiation software. Scale bars equal 10μm. (Bi) Fixed and permeabilized day-6 erythroblasts were dual stained with PrPc mAb (red) and CD82 mAb (green). Overlays were prepared and highlighted areas magnified to show (Bii) plasma membrane at cell-cell contact site and (Biii) internal PrP-containing vesicle (arrow). Scale bars equal 10 μm. Rebecca E. Griffiths et al. Blood 2007;110:4518-4525 ©2007 by American Society of Hematology

Patching experiments suggest CD81 and PrPc can share the same microdomain. Patching experiments suggest CD81 and PrPc can share the same microdomain. ROIs were drawn along the cell surface of each merge image, and profiles of green and red fluorescence along these ROI were constructed using Leica quantiation software. (Ai) Control cells were fixed then stained for PrP (red) and CD81 (green). (Aii) Cells underwent PrP antibody–induced patching (red) then fixed and dual stained for CD81 (green). Scale bars equal 10 μm. (Bi) Control cells were fixed then stained for CD81 (red) and PrP (green). (Bii) Cells underwent CD81 antibody-induced patching (red), then were fixed and dual stained for PrP (green). (Ci) Cells underwent PrP antibody–induced patching (red), then were fixed and dual stained for CD59 (green). (Cii) Cells underwent CD81 antibody–induced patching (red), then were fixed and then dual stained for CD59 (green). (Di) Cells underwent CD59 antibody–induced patching (red), then were fixed and dual stained for PrP (green). (Dii) Cells underwent CD59 antibody–induced patching (red), then were fixed and dual stained for CD81 (green). Rebecca E. Griffiths et al. Blood 2007;110:4518-4525 ©2007 by American Society of Hematology

A proposed model for the internalization of PrPc in erythroblasts. Rebecca E. Griffiths et al. Blood 2007;110:4518-4525 ©2007 by American Society of Hematology