Figure 4.
Caveolae-mediated uptake of PrP C is cholesterol dependent. (A) PrPC-dependent protein A–gold internalization in SHaPrPC- expressing CHO/30C3 cells was temperature sensitive. Cells were preincubated with protein A–gold (5 nm) at 4°C before fixation. Ultrathin cryosections were labeled with an antibody against caveolin-1 (10-nm gold). Abundant protein A–gold was only seen on the plasma membrane and on plasma membrane–associated caveolae (thick arrow). Caveolin-1–positive endocytic structures (arrowheads) and caveolae-1 intracellular caveolae (arrowheads) did not exhibit any protein A–gold labeling (compare with Fig. 5). (B) Effect of filipin treatment on protein A–gold uptake and the fate of caveolae. Cells were treated for 24 h with filipin followed by a 10-min incubation in media that also contained protein A–gold (5 nm) and a 50-min chase at 37°C. Sections were labeled with anti–caveolin-1 (15 nm). A significant number of protein A–gold (small size) binding on the membrane was seen but no enrichment in caveolae (arrow). The uptake of protein A–gold is sharply reduced; endocytic structures contain protein A–gold (small size) at relatively low levels. No evidence of caveolae and no label for caveolin-1 (large gold) can be seen on these endocytic structures. Many caveolin-1–positive (large gold) vesicles/tubules are present in the pericentriolar region devoid of small protein A–gold. c, centriole; e, endocytic structure; m, mitochondria; n, nucleus; p, plasma membrane. Bars, 200 nm.