Publication Type Journal Article
Title Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions
Authors Evandro Tavares Joana A. Macedo Pedro M. R. Paulo Catarina Tavares Carlos Lopes Eduardo P. Melo
Groups MPPM
Journal BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
Year 2014
Month July
Volume 1842
Number 7
Pages 981-991
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. (C) 2014 Elsevier B.V. All rights reserved.
DOI http://dx.doi.org/10.1016/j.bbadis.2014.02.002
ISBN
Publisher ELSEVIER SCIENCE BV
Book Title
ISSN 0925-4439
EISSN 0006-3002
Conference Name
Bibtex ID ISI:000336698400010
Observations
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