Measuring Protein Interactions Using Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy

Abstract

The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster Resonance Energy Transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/ EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Keywordsfluorescence lifetime imaging microscopy (FLIM); Förster resonance energy transfer (FRET) microscopy; fluorescent proteins (FPs); protein-protein interactions (PPI)

PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

“…The phasor plot shows that the lifetime is quenched compared to the lifetime for mTurquoise2 alone (compare with Fig. 2 A; Table S1), and the lifetime distribution is shifted inside the universal semicircle, indicating a multiexponential decay consistent with FRET (22). The average lifetime is 2.5 ns, corresponding to a FRET efficiency of 37% (Table S2).…”

“…MLO-Y4 osteocytes (21) were cultured on collagen-coated plates (rat tail collagen type I; BD Biosciences, San Jose, CA) in Minimum Essential Medium-a (Gibco, Life Technologies, Carlsbad, CA) supplemented with 5% FCS, 5% fetal bovine serum, and 1% penicillin/streptomycin (Gibco). The cells were harvested at 70-80% confluence and transfected by electroporation at 250 V with a 9-ms pulse duration using a BTX ECM 830 electroporator (Harvard Apparatus, Holliston, MA) as described earlier (22). The cells are immediately recovered from the cuvette and diluted in phenol red-free tissue culture medium containing serum.…”

Fast fluorescence lifetime imaging techniques: A review on challenge and development

“…Often the demand for fast FLIM just originates from the desire to¯nish a given experiment in a shorter period of time, in other cases, there is the need for dynamic imaging of fast moving cells and organelles, vesicle tra±cking in live cells, 25 and dynamic changes in ion concentrations, 26,27 metabolic state, 28,29 or interactions between proteins. 30,31 As a consequence, there is currently a run toward faster and faster FLIM techniques with a steeply increasing number of publications. There are FLIM techniques recording in the time domain and in the frequency domain, scanning techniques and wide-¯eld techniques, and analog-recording and photon counting techniques.…”

When the Complex Makes It Easy: Phasor Plotting as a Model Independent Representation of Fluorescence Decay in Flow Cytometry

“…of selectively detecting different types of dyes and their interactions, where possible contaminating effects of hardly controllable factors such as exciting light flux and local exciting dye concentration are absent (1,2). Time-and frequencydomain detections of fluorescence lifetime have been elaborated both in microscopy and flow cytometry (2)(3)(4)(5).…”