Understanding FRET as a Research Tool for Cellular Studies

Abstract

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

SIGMAR1 Regulates Membrane Electrical Activity in Response to Extracellular Matrix Stimulation to Drive Cancer Cell Invasiveness

“…These data strongly suggest that Sig1R is necessary to promote the hERG/b1-integrin interaction and that Sig1R itself is included within the complex. To further confirm this mechanism, we performed a flow cytometry-based FRET assay on nonpermeabilized cells to detect hERG/b1-integrin physical interaction at K562 cell surface (28)(29)(30). To assess a correct FRET signal, K562 cells stained with only one of both fluorochromes were used as control to determine the FRET-positive gate (FRET þ ).…”

“…Cells were analyzed with BD LSR Fortessa and FACS Diva software (Becton Dickinson) on the basis of 10,000 events. The methodology of flow cytometry-based F€ orster resonance energy transfer (FRET) assay was previously described (28)(29)(30). Briefly, the donor protein was stained with Alexafluor 488-conjugated antibody, and the acceptor protein was stained with Alexafluor 594-conjugated antibody.…”

Analysis of ER-mitochondria contacts by correlative fluorescence microscopy and soft X-ray tomography of mammalian cells

“…FRET detection and analysis was performed using acceptor photobleaching experiments (Shrestha et al, 2015). GFP and RFP and/or mCherry constructs of the respective fission proteins were co-transfected into cells.…”

Fluorescence Lifetime Imaging of a Caspase‐3 Apoptosis Reporter

“…Efficient FRET reduces fluorescence from the donor relative to the acceptor. Intensity-based FRET measurements are affected by relative concentrations of donor and acceptor molecules, and wavelength-dependent differences in attenuation and scattering of light also distort intensity ratios in tissues (Hoppe, Christensen, & Swanson, 2002; Shrestha, Jenei, Nagy, Vereb, & Szöllősi, 2015; Yellen & Mongeon, 2015). As an alternative to intensity-based measurements, we and others have used fluorescence lifetime imaging microscopy (FLIM) to quantify FRET.…”