title: Homodimerization of amyloid precursor protein at the plasma membrane: a homoFRET study by time-resolved fluorescence anisotropy imaging.
abstract: Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer's disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.
authors.affiliation: Institut des Sciences Moléculaires d'Orsay, CNRS UMR 8214, Univ. Paris Sud, Orsay, France.
authors.forename: Viviane
authors.initials: V
authors.lastname: Devauges
authors.forename: Catherine
authors.initials: C
authors.lastname: Marquer
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lécart
authors.forename: Jack-Christophe
authors.initials: JC
authors.lastname: Cossec
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Potier
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Fort
authors.forename: Klaus
authors.initials: K
authors.lastname: Suhling
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lévêque-Fort
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D005454:Fluorescence Polarization; D015200:Fluoroimmunoassay / Q000379:methods*; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D013050:Spectrometry, Fluorescence
chemical_list: D016564:Amyloid beta-Protein Precursor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins