Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells

M. P. McKinley, A. Taraboulos, L. Kenaga, D. Serban, A. Stieber, S. J. DeArmond, S. B. Prusiner*, N. Gonatas

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

195 Scopus citations

Abstract

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) designated PrP(Sc). In scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells, PrP(Sc) accumulates primarily within the cell cytoplasm, whereas cellular PrP (PrP(C)) is anchored to the external surface of the plasma membrane by a glycoinositol phospholipid moiety. To determine the subcellular localization of PrP(Sc), scrapie-infected cells were grown to ~75% confluency, fixed briefly, and then incubated with guanidine thiocyanate before antibody staining and examination by electron microscopy. PrP(Sc) immunoreactivity was enhanced by denaturation with guanidine isothiocyanate which also permeabilized cells (Taraboulos et al., J Cell Biol 110:2117, 1990). As judged both by deposition of immunoperoxidase reaction product (diaminobenzidine) and by presence of immunogold particles, PrP(Sc) was identified in discrete vesicular foci and some large bodies in the cytoplasm of scrapie-infected cells. Some vesicles with PrP(Sc) staining also contained myelin figures resembling those found in autophagic vacuoles forming secondary lysosomes. The presence of PrP(Sc) in secondary lysosomes is inferred from colocalization of guanidine isothiocyanate enhanced PrP immunoreactivity and acid phosphatase. Neither the diaminobenzidine reaction product nor immunogold particles were observed in association with the nucleus, endoplasmic reticulum, or Golgi stacks. Exposure of scrapie-infected cells to the brefeldin A dispersed the Golgi apparatus but did not alter the morphologic distribution of PrP(Sc), indicating that no detectable PrP(Sc) was associated with Golgi stacks. It remains to be established whether secondary lysosomes are involved in the post-translational formation of PrP(Sc).

Original languageEnglish
Pages (from-to)622-630
Number of pages9
JournalLaboratory Investigation
Volume65
Issue number6
StatePublished - 1991
Externally publishedYes

Keywords

  • Autophagic vacuoles
  • Brefeldin A
  • Immunocytochemistry
  • Protein denaturation
  • Secondary lysosomes

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