Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions

Abstract

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET

“…For example, a study of GPI-anchored 5’-nucleotidase distribution on the plasma membrane using FRET imaging followed labeling with fluorophore-conjugated monovalent Fab and the bivalent IgG antibody [ 8 ]. There are examples of the successful generation of functional fusions of GPI-AP with FP that can be used for FRET studies too [ 21 , 22 ].…”

“…First, we inserted monomeric teal FP 1 (mTFP1 or TFP) as a donor and yellow fluorescent protein (tagYFP or YFP) as an acceptor near the T-cadherin GPI-attachment site [ 20 ], the same site used in some other research on GPI-APs [ 21 , 22 ]. Briefly, sequences that encoded the fluorescent proteins YFP or TFP, with the addition of linkers on each side and without stop codons, were inserted between the DNA sequences that coded the fifth cadherin motif and GPI-anchoring signal in human full-length T-cadherin cDNA: Leu675-His-Gly-FP-Glu-His-Ala-Arg676.…”

Membrane-enriched proteome changes and prion protein expression during neural differentiation and in neuroblastoma cells

“…The levels of PrP in PK1-KD cells are almost undetectable by western blot and immunocytochemistry [21, 40]. From a selection of 41 spots with significantly different abundance, 23 were picked resulting in the identification of 8 spots and 6 distinct proteins (Fig.…”

“…Quantitative 2D-DIGE was also performed for comparison between PK1 neuroblastoma cells expressing PrP and PK1-KD which are knockdown for PrP, aiming at gathering information about the effect of PrP ablation on the membrane enriched proteome. The levels of PrP in PK1-KD cells are almost undetectable by western blot and immunocytochemistry [ 21 , 40 ]. From a selection of 41 spots with significantly different abundance, 23 were picked resulting in the identification of 8 spots and 6 distinct proteins (Fig.…”

“…ATP5A1 increased upon neural differentiation, concomitantly with the levels of PrP, but ATP5B also increased significantly in PK1-KD cells. PK1 cells, which are highly efficient in the replication of RML prions [ 26 ], were infected as previously described [ 40 ] and a 2D-DIGE experiment comparing non-infected and infected cells was carried out. This experiment gave no significant differential abundance of proteins (data not shown), which was not surprising since no transcriptional changes seem to be induced by prion infection of neural cell lines [ 41 ].…”