Multi-color fluorescence fluctuation spectroscopy detects higher-order molecular interactions in living cells

Session

Using FLIM and FCS to Determine Interactions and Dynamics

Authors

Dr. Valentin Dunsing (1, 2), Annett Petrich (1), Prof. Salvatore Chiantia (1)

Affiliations

1. University of Potsdam
2. Aix-Marseille University/CNRS

Keywords

Fluorescence Microscopy, Fluorescence Fluctuation Spectroscopy, Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, Plasma Membrane, Protein-Protein Interactions, Diffusion, Oligomerization, Biomolecular Interactions, Morphogenesis

Abstract text

Living cells rely on transport and interaction of biomolecules to perform their diverse functions. To obtain a detailed understanding of these highly dynamic processes in the native environment, minimally invasive techniques are needed. A powerful toolbox of such techniques is provided by fluorescence fluctuation spectroscopy (FFS). In more detail, FFS takes advantage of the inherent dynamics present in biological systems, such as diffusion, to infer molecular parameters from fluctuations of the signal emitted by an ensemble of fluorescently tagged molecules. In my talk, I will introduce the conceptual principle of FFS and several FFS variants, such as fluorescence cross-correlation spectroscopy (FCCS). I will then present our recent efforts on 1) how to accurately quantify the stoichiometry of protein complexes in living cells by taking dark states of fluorescent tags into account [1], 2) how to multiplex FFS measurements using spectral detection to probe diffusion and higher order interactions of up to four molecular species simultaneously [2], 3) applying FFS in biological context, e.g. studying the assembly process of Influenza viruses at the plasma membrane of infected host cells [2,3]. Finally, I will showcase the power of FFS techniques for studying highly dynamic morphogenetic processes in living embryos.

References

[1] Dunsing, V., Luckner, M., Zühlke, B. et al. Optimal fluorescent protein tags for quantifying protein oligomerization in living cells. Sci Rep 8, 10634 (2018)

[2] Valentin Dunsing, Annett Petrich, Salvatore Chiantia (2021) Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection. eLife 10:e69687

[3] Annett Petrich, Valentin Dunsing, Sara Bobone, Salvatore Chiantia Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study. Biophysical Journal 120, 5478–5490, 2021