Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins

Abstract

Fluorescence fluctuation spectroscopy (FFS) can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis (SpIDA) and fluorescence moment image analysis (FMIA) are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color SpIDA (2c-SpIDA) and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering (SSF) technique to remove artifacts due to intrinsic crosstalk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species, hence this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with EGFP and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments. Statement of SignificanceFluorescence fluctuation spectroscopy (FFS) techniques determine biophysical parameters from samples containing fluorescently labeled biomolecules by considering the statistical nature of fluorescent signals measured with photodetectors. The present study introduces two-color spatial cumulant analysis (2c-SpCA) to the canon of FFS techniques. 2c-SpCA analyzes pixel-value data of two-color images collected with laser scanning fluorescence microscopes. We show that 2c-SpCA can determine several biophysical parameters in living cells including Forster resonance energy transfer efficiency, the dark state fraction of fluorescent proteins, and heteromerization between distinctly labeled proteins. In comparison to existing techniques, 2c-SpCA requires very few image frames for analysis, minimal computations, and can be applied to images of fixed tissue samples.

Enhancing neuronal chloride extrusion rescues α2/α3 GABAA-mediated analgesia in neuropathic pain

“…Spatial intensity distribution analysis. SpIDA is an analysis technique that was developed to quantify complex mixtures of oligomerization states using fluorescent microscopy images 36,133,141 . SpIDA can measure the concentration and oligomeric states of proteins revealed with fluorescent probes (e.g., fluorescent proteins, fluorescent antibodies).…”

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

“…Next, we tested whether the same approach can be used for FPs with overlapping emission in the red region of the visible spectrum, which generally suffer from reduced SNR in FFS applications (14, 30). Therefore, we performed SFSCS measurements on HEK 293T cells co-expressing mp-mCherry2 and mp-mApple.…”

“…Notably, two-species SFSCS could not only successfully discriminate between mEGFP and mEYFP, but was also applicable on the red FPs mApple and mCherry2. These two FPs were successfully used in several FFS studies (14, 30, 39), providing the best compromise between brightness, maturation and photostability among available red FPs, which generally suffer from reduced SNR compared to FPs emitting in the green or yellow part of the optical spectrum (13, 14, 40).…”

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

“…Notably, two-species SFSCS can not only successfully discriminate between mEGFP and mEYFP, but is also applicable when using the red FPs mApple and mCherry2. These two FPs were successfully used in several FFS studies (14,30,39), providing the best compromise between brightness, maturation and photostability among available red FPs, which generally suffer from reduced SNR compared to FPs emitting in the green or yellow part of the optical spectrum (13,14,40).…”

“…Next, we tested whether the same approach can be used for FPs with overlapping emission in the red region of the visible spectrum, which generally suffer from reduced SNR in FFS applications (14,30). Therefore, we performed SFSCS measurements on HEK 293T cells coexpressing mp-mCherry2 and mp-mApple.…”