Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

作者：Devauges Viviane^*; Marquer Catherine; Lecart Sandrine; Cossec Jack Christophe; Potier Marie Claude; Fort Emmanuel; Suhling Klaus; Leveque Fort Sandrine

来源：PLos One, 2012, 7(9): e44434.

DOI：10.1371/journal.pone.0044434

Classical FRET (Forster 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%26apos;s disease, was measured on this original setup. 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.

• 出版日期2012-9-4