Observing fibrillar assemblies on scrapie-infected cells

Abstract

The infectious agent in prion diseases is an aberrant-folded isoform of the cellular prion protein (PrP C ). This scrapie-related prion protein (PrP Sc ) has an increased β-sheet content, is detergent insoluble and proteinase K resistant, and accumulates in prion-infected organisms and cells. In vitro, PrP Sc self-aggregates into amyloid fibrils. However, there is no direct experimental proof for the occurrence of PrP Sc -containing fibrils in vivo or in cell cultures. Applying atomic force microscopy (AFM) to scrapie-infected mouse neuroblastoma (ScN2a) cells, we discovered growing patch-like assemblies of amyloid-like fibrillar structures on the cell surfaces. Immunofluorescence and AFM images showed heterogeneous accumulation and aggregation of PrP Sc in ScN2a cell cultures. The percentage of cells having characteristic fibrils on their surface increased with time after scrapie infection. These endogeneous fibrils had lengths from 0.5 to 3 μm and protruded from the cell surface by 108±30 nm, and thus resembled the heterogeneous shapes and networks of in vitro prepared amyloid fibrils.

Progress towards structural understanding of infectious sheep PrP-amyloid

“…9 In contrast, ovine PrP Sc -fibrils Sc in living cells which was observed by light, atomic force, and scanning electron microscopy to form thin, 5 mm-long structures on the cell surface. 36,37 Regarding the average width of PrP Sc -fibrils, their appearance in our AFM images was obscured by co-purified brain matter in the form of extraneous non-fibrillar material. Also the natural presence of GPI-anchor and un-, mono, and diglycosylated PrP Sc forms obscured the underlying proteinaceous parts of PrP Sc -fibrils causing high width standard deviations.…”

Getting a Grip on Prions: Oligomers, Amyloids, and Pathological Membrane Interactions

“…Multivalent membrane interactions should limit the quaternary interactions between PrP RES molecules within the plane of the membrane and may promote sheet-like lateral structures (125) compatible with the 2-D PrP RES crystals visualized by Wille et al (45). On the other hand, whole-cell atomic force microscopy suggests that fibrillar PrP RES aggregates might be present on the surface of scrapie-infected cells in culture (126). …”

Structural Insights into Alternate Aggregated Prion Protein Forms

“…AFM provided quantitative data that could enable the classification of different protein structures. [25][26][27][28][29][30] Hence, our study offers novel molecular insights into the complex assembly pathways acquired by recPrP.…”