Figure 3.
Strings are cell surface structures. (A) Unlike most PrP27-30, FL PrPSc strings do not reside in dextran-positive endosomes. ScGT1 were incubated with a fixable Alexa 555–dextran (2 h; 250 µg/ml; red) to saturate the endosomal system, fixed, permeabilized, denatured with 3 M GdnSCN, and stained with either core D13 (a–c) or N-terminal mAb 8B4 (d–f). Within the cell (a–c), most D13 colocalized with dextran (c), consistent with endocytic PrP27-30 localization. The dorsal z = 0 plane (d) contains strings but no dextran. Internalized dextran was first seen in the contiguous z = 300 nm plane within the cell interior (e). The two images are superposed in f. Images sharpened by 2D nearest neighbor deconvolution. (B) In chilled ScGT1, strings are found exclusively on the cell surface. (Step 1) Live ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fabs (red). (Step 2) Cells were fixed and permeabilized, blocked with unlabeled anti-mouse Fabs, and restained with 8B4 followed by a secondary IgG (green). No additional strings structures were observed in the green (c) versus the red (b) channels, indicating no intracellular strings.