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, micrometer-long 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 membrane-anchored PrP(Sc) amyloids.
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Alexander
A
Rouvinski
Sharon
S
Karniely
Maria
M
Kounin
Sanaa
S
Moussa
Miri D
MD
Goldberg
Gabriela
G
Warburg
Roman
R
Lyakhovetsky
Dulce
D
Papy-Garcia
Janine
J
Kutzsche
Carsten
C
Korth
George A
GA
Carlson
Susan F
SF
Godsave
Peter J
PJ
Peters
Katarina
K
Luhr
Krister
K
Kristensson
Albert
A
Taraboulos
D000199:Actins / Q000378:metabolism; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D052160:Benzothiazoles; D002470:Cell Survival; D004705:Endocytosis; D006624:Hippocampus / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D013194:Staining and Labeling; D013844:Thiazoles / Q000378:metabolism
D000199:Actins; D000682:Amyloid; D000906:Antibodies; D052160:Benzothiazoles; D011134:Polysaccharides; D018620:PrPSc Proteins; D013844:Thiazoles; C009462:thioflavin T; D054801:Phosphoinositide Phospholipase C