title: 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.
authors: Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors: Evandro
authors: E
authors: Tavares
authors: Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors: Joana A
authors: JA
authors: Macedo
authors: Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal.
authors: Pedro M R
authors: PM
authors: Paulo
authors: Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors: Catarina
authors: C
authors: Tavares
authors: Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors: Carlos
authors: C
authors: Lopes
authors: Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal; Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal. Electronic address: emelo@ualg.pt.
authors: Eduardo P
authors: EP
authors: Melo
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011328:Prions; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: CFP-YFP; FLIM; Live-cell FRET imaging; Prion propagation; Prion protein; m-turquoise2