TY - JOUR
T1 - Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells
AU - McKinley, M. P.
AU - Taraboulos, A.
AU - Kenaga, L.
AU - Serban, D.
AU - Stieber, A.
AU - DeArmond, S. J.
AU - Prusiner, S. B.
AU - Gonatas, N.
PY - 1991
Y1 - 1991
N2 - 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).
AB - 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).
KW - Autophagic vacuoles
KW - Brefeldin A
KW - Immunocytochemistry
KW - Protein denaturation
KW - Secondary lysosomes
UR - http://www.scopus.com/inward/record.url?scp=0026326210&partnerID=8YFLogxK
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C2 - 1684401
AN - SCOPUS:0026326210
SN - 0023-6837
VL - 65
SP - 622
EP - 630
JO - Laboratory Investigation
JF - Laboratory Investigation
IS - 6
ER -