Abstract
We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with Förster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic "fingerprint" of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known "nanoruler" to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings. Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled β2-adrenergic receptor (β2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize. Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.
Highlights
Fluorescence spectroscopy is one of the main methods to quantify protein dynamics and protein-protein interactions with minimal perturbation in a cellular context
When combined with Förster resonance energy transfer (FRET), intramolecular dynamics such as conformational changes can be monitored at the same time in a common confocal setup[5, 6]
Calibration Measurements NOTE: The FRET-fluorescence correlation spectroscopy (FCS) setup is equipped with a confocal microscope water objective, two laser lines, a Time-Correlated Single Photon Counting (TCSPC) system, two hybrid photomultiplier tubes (PMT) and two avalanche photodiodes (APD) for photon collection and the data collection software
Summary
The main underlying principle in correlation spectroscopy is the statistical analysis of intensity fluctuations emitted by fluorescently labeled biomolecules diffusing in and out of a laser focus (Figure 1A). Jove.com fluorophores are on the same side of the cell membrane (CTSNAP) It carries a C-terminal SNAP-tag and an intracellular eGFP. Again both labels are at the same protein with again 100% co-diffusion expected. Simulated data exemplifies the influence of experimental parameters on the FRET-induced anticorrelation, and the effect of protein-protein interactions on the co-diffusion amplitude. This protocol provides a complete guide to performing the combined FRET-FCCS in living cells to understand protein dynamics and protein-protein interactions while making aware of technical/physical artifacts, challenges, and possible solutions
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