Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

Mammalian prions refold host glycosylphosphatidylinositol- anchored PrP ^C into β-sheet- rich PrP ^Sc. PrP ^Sc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP ^Sc rather than on its truncated PrP27-30 product. We show that N-terminal PrP ^Sc epitopes are exposed in their physiological context and visualize, for the first time, PrP ^Sc in living cells. PrP ^Sc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometerlong structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membraneanchored PrP ^Sc amyloids.