Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
- Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopySignaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.…
Author details: | Valentin DunsingORCiDGND, Annett PetrichORCiDGND, Salvatore ChiantiaORCiDGND |
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DOI: | https://doi.org/10.7554/eLife.69687 |
ISSN: | 2050-084X |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/34494547 |
Title of parent work (English): | eLife |
Publisher: | eLife Sciences Publications |
Place of publishing: | Cambridge |
Publication type: | Article |
Language: | English |
Year of first publication: | 2021 |
Publication year: | 2021 |
Release date: | 2023/01/05 |
Tag: | Viruses; diffusion; fluorescence; interactions; optical microscopy; protein-protein; virus assembly |
Volume: | 10 |
Article number: | e69687 |
Number of pages: | 33 |
Funding institution: | German Research Foundation (DFG)German Research Foundation (DFG) [254850309]; DFGGerman Research Foundation (DFG)European Commission [INST 336/114-1 FUGG] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 50 Naturwissenschaften / 500 Naturwissenschaften und Mathematik |
6 Technik, Medizin, angewandte Wissenschaften / 60 Technik / 600 Technik, Technologie | |
Peer review: | Referiert |
License (German): | CC-BY - Namensnennung 4.0 International |