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Rapid cell-surface prion protein conversion revealed using a novel cell system.

Goold R, Rabbanian S, Sutton L, Andre R, Arora P, Moonga J, Clarke AR, Schiavo G, Jat P, Collinge J, Tabrizi SJ - Nat Commun (2011)

Bottom Line: Prion diseases are fatal neurodegenerative disorders with unique transmissible properties.Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line.The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrP(C)). Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)). Using this epitope-tagged PrP(Sc), we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.

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Related in: MedlinePlus

Prion conversion first occurs at the cell surface within 1 min of prion exposure.(a) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrPSc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrPSc also shows low intracellular levels of PrPSc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrPSc, with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. (b) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; *P<0.05, **P<0.01, two-tailed t-test. The exact P-values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). (c) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrPSc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.
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f4: Prion conversion first occurs at the cell surface within 1 min of prion exposure.(a) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrPSc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrPSc also shows low intracellular levels of PrPSc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrPSc, with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. (b) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; *P<0.05, **P<0.01, two-tailed t-test. The exact P-values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). (c) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrPSc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.

Mentions: The steep rise in the number of prion-infected cells observed in the first 2 min following RML prion exposure indicates that this is the period when most cells first begin to synthesize PrPSc (Fig. 3c). This allowed us to study the cellular site of this initial prion conversion. Examination of cells that were fixed after 1 min of prion exposure revealed that the MYC-tagged PrPSc signal is only present at the plasma membrane. Orthogonal projections of serial confocal sections clearly showed that PrPSc is present only at the cell surface (Fig. 4a—1 min, white arrows—the z stack series is shown in Supplementary Fig. S5a). This cellular distribution is strikingly different from the more typical plasma membrane and intracellular localization observed in cells that have been exposed to RML prions for longer durations (Fig. 4a—180 min, arrows—the z stack series is shown in Supplementary Fig. S5b). Categorizing the cells into three phenotypes of different PrPSc distributions (plasma membrane, plasma membrane/diffuse intracellular and plasma membrane/PNC), and examining each phenotype at different durations following RML prion exposure revealed a rapid transition from the plasma membrane distribution seen at the earliest time point to the typical plasma membrane/PNC distribution observed later (Fig. 4b). Indeed, cells showing exclusively plasma membrane MYC-tagged PrPSc staining disappeared after 2 min of exposure to RML prions, consistent with rapid PrPSc formation, endocytosis and trafficking.


Rapid cell-surface prion protein conversion revealed using a novel cell system.

Goold R, Rabbanian S, Sutton L, Andre R, Arora P, Moonga J, Clarke AR, Schiavo G, Jat P, Collinge J, Tabrizi SJ - Nat Commun (2011)

Prion conversion first occurs at the cell surface within 1 min of prion exposure.(a) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrPSc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrPSc also shows low intracellular levels of PrPSc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrPSc, with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. (b) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; *P<0.05, **P<0.01, two-tailed t-test. The exact P-values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). (c) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrPSc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3104518&req=5

f4: Prion conversion first occurs at the cell surface within 1 min of prion exposure.(a) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrPSc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrPSc also shows low intracellular levels of PrPSc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrPSc, with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. (b) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; *P<0.05, **P<0.01, two-tailed t-test. The exact P-values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). (c) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrPSc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.
Mentions: The steep rise in the number of prion-infected cells observed in the first 2 min following RML prion exposure indicates that this is the period when most cells first begin to synthesize PrPSc (Fig. 3c). This allowed us to study the cellular site of this initial prion conversion. Examination of cells that were fixed after 1 min of prion exposure revealed that the MYC-tagged PrPSc signal is only present at the plasma membrane. Orthogonal projections of serial confocal sections clearly showed that PrPSc is present only at the cell surface (Fig. 4a—1 min, white arrows—the z stack series is shown in Supplementary Fig. S5a). This cellular distribution is strikingly different from the more typical plasma membrane and intracellular localization observed in cells that have been exposed to RML prions for longer durations (Fig. 4a—180 min, arrows—the z stack series is shown in Supplementary Fig. S5b). Categorizing the cells into three phenotypes of different PrPSc distributions (plasma membrane, plasma membrane/diffuse intracellular and plasma membrane/PNC), and examining each phenotype at different durations following RML prion exposure revealed a rapid transition from the plasma membrane distribution seen at the earliest time point to the typical plasma membrane/PNC distribution observed later (Fig. 4b). Indeed, cells showing exclusively plasma membrane MYC-tagged PrPSc staining disappeared after 2 min of exposure to RML prions, consistent with rapid PrPSc formation, endocytosis and trafficking.

Bottom Line: Prion diseases are fatal neurodegenerative disorders with unique transmissible properties.Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line.The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrP(C)). Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)). Using this epitope-tagged PrP(Sc), we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.

Show MeSH
Related in: MedlinePlus