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

J Cell Biol. 2014 Feb 3;204(3):423-41. doi: 10.1083/jcb.201308028.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Amyloid / chemistry
  • Amyloid / metabolism*
  • Amyloid / ultrastructure
  • Animals
  • Antibodies / metabolism
  • Benzothiazoles
  • Cell Survival
  • Endocytosis
  • Hippocampus / metabolism
  • Imaging, Three-Dimensional*
  • Membrane Microdomains / metabolism*
  • Mice
  • Models, Biological
  • Phosphoinositide Phospholipase C / metabolism
  • Polysaccharides / metabolism
  • PrPSc Proteins / chemistry
  • PrPSc Proteins / metabolism*
  • Protein Binding
  • Protein Denaturation
  • Staining and Labeling
  • Thiazoles / metabolism

Substances

  • Actins
  • Amyloid
  • Antibodies
  • Benzothiazoles
  • Polysaccharides
  • PrPSc Proteins
  • Thiazoles
  • thioflavin T
  • Phosphoinositide Phospholipase C