Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

Abstract

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 inter-molecular 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.

Mandipropamid as a chemical inducer of proximity for in vivo applications

“…To further characterize the interaction efficiency between receiver and receptor domains in the new Mandi CIP system, we measured the relative amount of receiver bound to receptor in the absence and presence of CIPs using raster image correlation spectroscopy (RICS) 20 . After transient expression of cytosolic receiver and receptor domains fused to spectrally different fluorescent proteins, we determined the interacting fraction by computing the cross-correlation functions (CCF) between spectral channels and normalizing the obtained CCF amplitudes to controls with or without constitutive interaction between fluorophores 21 (Extended Data Fig.…”

“…RSICS analysis followed the implementation described recently 39 , 47 , which is based on applying the mathematical framework of fluorescence lifetime and fluorescence spectral correlation spectroscopy 48 , 49 to RICS. Four-dimensional image stacks were imported in MATLAB (The MathWorks) from CZI image files using the Bioformats package 49 and further analyzed using custom-written code.…”

Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

“…For example, our control experiments showed that the cellular distribution of M1 with an mEGFP fused to its C-terminus was similar to that of unlabeled M1 (46,47), whereas an N-terminally fused mEGFP M1 variant seemed to have transport failures which are probably caused by steric hindrance between fluorophore and signal peptide (47). On the other hand, the fluorescent constructs used to investigate the viral envelope proteins (HA, NA, and M2) were all localized at the PM, similar to the corresponding non-fluorescent proteins (48,49), and yielded the expected oligomerization state (41,42,53,54). For example, our results are compatible with the presence of NA tetramers and mixtures of M2 dimers and tetramers (Figure 3 C), in agreement with previous data (55,70).…”

“…To this aim, we selected HEK293T cells as a cellular model because they are often used for reverse genetic virus production (39,40,67) and were shown to be appropriate for IAV protein expression (17,41,42). Additionally, we produced and tested several fluorescent IAV protein constructs.…”

“…The plasmids encoding the fluorescence proteins (FP) mEGFP or mCherry2 linked to a myristoylated and palmitoylated peptide (mp-mEGFP, mp-mCherry2, mp-2x-mEGFP), and the plasmids for cytosolic expression of mEGFP, 2x-mEGFP were previously described (41) and are available on Addgene (Watertown, MA, USA). The plasmids encoding the FP hetero-dimer mCherry2-mEGFP linked to a myristoylated and palmitoylated peptide (mp-mCherry2-mEGFP), and the matrix protein 2 (M2) of FPV with mCherry2 fused to the extracellular terminus of M2 (mCherry2-M2) were previously described (42). Further information regarding other plasmids and constructs used in this work is provided in the Supplemental Information (SI).…”