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.
Highlights
Protein interactions are central to most of physiological and pathological processes and studying them dynamically is one of the challenges of current biology
We present the implementation of time-resolved Fluorescence Anisotropy IMaging on a Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) setup to study protein homodimerization at the plasma membrane of living cells
We considered the measurements made with the objective 106 as our reference and corrected the fluorescence anisotropy decay obtained with the objective 606 . xNA can be deduced from the formula below obtained from re-arranging Eq.4 : xNA
Summary
Protein interactions are central to most of physiological and pathological processes and studying them dynamically is one of the challenges of current biology. FRET imaging is an sensitive and widely used technique to monitor proteins interactions in living cells [1]. FRET can be measured by Fluorescence Lifetime Imaging Microscopy (FLIM). As FLIM is independent of fluorophore concentration and can be used to monitor small changes in fluorescence lifetime, it is currently considered the most accurate technique to probe protein heterodimerization [6]. This technique of classical FRET using two fluorescent labels (one for the donor and another one for the acceptor) is less efficient for studying the homodimerization or multimerization of a protein. Only half of the homodimers formed can be identified by this technique
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