Ultrastructural Localization of Scrapie Prion Proteins in Cytoplasmic Vesicles of Infected Cultured Cells

Lab Invest. 1991 Dec;65(6):622-30.

Abstract

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) designated PrPSc. In scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells, PrPSc accumulates primarily within the cell cytoplasm, whereas cellular PrP (PrPC) is anchored to the external surface of the plasma membrane by a glycoinositol phospholipid moiety. To determine the subcellular localization of PrPSc, scrapie-infected cells were grown to approximately 75% confluency, fixed briefly, and then incubated with guanidine thiocyanate before antibody staining and examination by electron microscopy. PrPSc 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, PrPSc was identified in discrete vesicular foci and some large bodies in the cytoplasm of scrapie-infected cells. Some vesicles with PrPSc staining also contained myelin figures resembling those found in autophagic vacuoles forming secondary lysosomes. The presence of PrPSc 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 PrPSc, indicating that no detectable PrPSc was associated with Golgi stacks. It remains to be established whether secondary lysosomes are involved in the post-translational formation of PrPSc.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acid Phosphatase / analysis
  • Animals
  • Brain Chemistry
  • Cells, Cultured
  • Cricetinae
  • Cytoplasm / chemistry*
  • Guanidines / pharmacology
  • Immunohistochemistry
  • Lysosomes / chemistry
  • Mice
  • Microscopy, Electron
  • PrPSc Proteins
  • Prions / analysis*
  • Prions / immunology
  • Rabbits
  • Scrapie / metabolism*
  • Thiocyanates / pharmacology

Substances

  • Guanidines
  • PrPSc Proteins
  • Prions
  • Thiocyanates
  • guanidine thiocyanate
  • Acid Phosphatase