Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Hemmen K, Choudhury S, Friedrich M, Balkenhol J, Knote F, Lohse MJ, Heinze KG (2021)
Publication Type: Journal article
Publication year: 2021
Journal
Book Volume: 2021
Article Number: e62954
Journal Issue: 178
DOI: 10.3791/62954
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.
Involved external institutions
Julius-Maximilians-Universität Würzburg
Germany (DE)
Max-Delbrück-Centrum für Molekulare Medizin / Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch
Germany (DE)
Germany (DE)
Max-Delbrück-Centrum für Molekulare Medizin / Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch
Germany (DE)
How to cite
APA:
Hemmen, K., Choudhury, S., Friedrich, M., Balkenhol, J., Knote, F., Lohse, M.J., & Heinze, K.G. (2021). Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells. Journal of Visualized Experiments, 2021(178). https://doi.org/10.3791/62954
MLA:
Hemmen, Katherina, et al. "Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells." Journal of Visualized Experiments 2021.178 (2021).
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