Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

Abstract

Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

Measuring G Protein Activation with Spectrally Resolved Fluorescence Fluctuation Spectroscopy

“…Ternary complex detection has been achieved with triple correlation analysis (TRICS), but that relies on higher order correlation functions which greatly increase the signal-to-noise requirements for the data and computational complexity (17,38). Additionally, visual inspection of covariance matrices allows for the straightforward observation of complex formation that has an intuitive connection to the emission spectra (Fig.…”

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

“…To this aim, we selected HEK293T cells as a cellular model because they are often used for reverse genetic virus production [40, 41, 79], were shown to be appropriate for IAV protein expression [17, 42, 43] and are better suited for quantitative fluorescence fluctuation analysis of proteins at the PM, compared to other cell lines [80]. Additionally, we produced and tested several fluorescent IAV protein constructs.…”

“…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 [44, 63], 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 [44]. 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 [65, 66], and yielded the expected oligomerization state [42, 43, 69, 70]. For example, our results are compatible with the presence of NA tetramers and mixtures of M2 dimers and tetramers (Figure 3C), in agreement with previous data [71, 83].…”

“…M1-M2 complexes appear to consist, in average, of 1-2 M1 and 2-4 M2 molecules (Figure 3). Assuming that each M2 monomer has a binding site for M1, the observed 1:2 stoichiometry suggests that the M1 binding might be limited for example by steric constraints or competition with other binding partners of M2 (e.g., LC3 [43]). Furthermore, in the simple approximation of M1 dimers, M2 tetramers, and 2:4 M1-M2 complexes being associated to the PM, our cross-correlation measurements indicate that ca.…”

“…The plasmids encoding the fluorescence proteins (FP) EGFP 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 [42] 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 [43].…”