Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.
diffusion
fluorescence
molecular biophysics
optical microscopy
physics of living systems
protein-protein interactions
structural biology
virus assembly
viruses
Journal
eLife
ISSN: 2050-084X
Titre abrégé: Elife
Pays: England
ID NLM: 101579614
Informations de publication
Date de publication:
08 09 2021
08 09 2021
Historique:
received:
23
04
2021
accepted:
07
09
2021
pubmed:
9
9
2021
medline:
15
12
2021
entrez:
8
9
2021
Statut:
epublish
Résumé
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 spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.
Identifiants
pubmed: 34494547
doi: 10.7554/eLife.69687
pii: 69687
pmc: PMC8545396
doi:
pii:
Substances chimiques
Fluorescent Dyes
0
M2 protein, Influenza A virus
0
Viral Matrix Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2021, Dunsing et al.
Déclaration de conflit d'intérêts
VD, AP, SC No competing interests declared
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