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

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 (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, 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.


Supplementary Figure S2 Comparison of the methods to visualise PrP Sc in RML prion infected cells. a) Uninfected PK1 cells (top panels) or iPK1 cells (from chronically RML prion infected
cultures, bottom panels) were fixed and treated with 98 % formic acid, proteinase K (PK) or guanidinium hydrochloride (GdnHCl) prior to staining with anti-PrP antibodies (green) and counter staining with DAPI (blue). Merged confocal images are shown; scale bar 20 µm. Uninfected cells show no staining after formic acid and PK treatment indicating that the PrP C has been removed. Some residual staining was observed after GdnHCl treatment. RML prion infected cells contain formic acid and PK resistant PrP (PrP Sc ) with a characteristic plasma membrane/perinuclear compartment distribution (arrows). b) Quantification of randomly chosen cell fields after the indicated treatments.
Quantification of randomly chosen cell fields from uninfected PK1 cells (blue bars) or chronically RML prion infected ScPK1 cells (red bars). Approximately half of the RML prion infected PK1 cells contained PrP Sc . The low level of background staining observed in the PK1 cells after formic acid and PK treatment is mostly attributable to clumped cells where access to the PrP C is restricted. GdnHCl treatment was less effective at removing PrP C staining. The mean ± SEM from three independent experiments are shown.

Supplementary Figure S3 Steady state subcellular distribution of MYC-tagged PrP Sc
Chronically RML prion infected PrP-224AlaMYC cells were fixed and treated with 98 % formic acid prior to staining with anti-MYC antibodies (green) and marker antibodies for various cell compartments as indicated (red); the cells were counter stained with DAPI.
Single channels and merged confocal images are shown as indicated; scale bar 20 µm.

PrP-224AlaMYC cells a) Short RML prion exposure is sufficient to generate cells that continue to
propagate PrP Sc in the absence of RML prion seed. PrP-224AlaMYC cells were b) Cells exposed to RML prions for 180 minutes were fixed and treated as in (a). Serial confocal sections show the typical distribution of PrP Sc at steady state, with strong immunostaining at the plasma membrane and in the perinuclear region; scale bar 10 µm.
The z stack series was used to generate the orthogonal projection shown in Fig. 4a (180 min).

Supplementary Figure S6 PrP Sc forms at the cell surface.
Pre-cooled PrP-224AlaMYC cells were exposed to RML prions on ice for two minutes then fixed and formic acid extracted prior to staining with anti-MYC antibodies (green) and counter staining with DAPI (blue). Serial confocal sections are shown. The z stack series was used to generate the orthogonal projection shown in Fig 5b; scale bar 20 µm. this is therefore PrP Sc derived from the inocula. In the upper panel one of the cells shows particularly strong anti-PrP labelling at the plasma membrane (white arrow) while in the lower panel a cell with strong intracellular anti-PrP labelling is highlighted (white arrow).
Despite this close association with high levels of inocula derived PrP Sc these cells do not immunostain with anti-MYC antibodies (green) and therefore have not generated MYCtagged PrP Sc . An adjacent cell in each cell field (yellow arrow) is labelled with both anti-PrP antibodies and anti-MYC antibodies showing it has generated de novo MYC tagged PrP Sc -these appear yellow in the merged image. This suggests that factors in addition to a simple physical association of cellular PrP C with PrP Sc affect prion conversion, an observation in agreement with previously published data.