N-Color Spatial Cumulant Analysis to Detect G-Protein Dynamics with Two-Photon Microscopy

Abstract

Brightness analysis in fluorescence fluctuation spectroscopy (FFS) is a tool used to measure the oligomerization of biomolecules carrying fluorescent labels. Conventionally, instruments for multicolor FFS experiments are configured so that there is one detector per chromophore. We utilize a multi-anode photon multiplier tube with many detection channels to achieve greater sensitivity by implementing n-color spatial cumulant analysis (NC-SpCA). This approach considers the multi-detector photon counting statistics of homogenous subcellular regions in fluorescence images to determine the concentrations and brightnesses of fluorescent species within the sample. Using NC-SpCA, we can, in principle, detect interactions in samples containing an arbitrary number of spectrally distinct chromophores. Canonically, trimeric G-protein complexes are composed of Gα, Gβ, and Gγ subunits. Ligand-activated GPCRs catalyze the dissociation of the complex into two signaling units: Gα and Gβγ. These units can participate in a myriad of downstream signaling pathways to regulate cellular behavior. We have created a single DNA construct for the expression of all three components of the trimeric G-protein complex tagged with spectrally distinct chromophores (mEGFP, mEYFP, mCherry2) in live cells. We use this tool in conjunction with NC-SpCA to measure activation of the G-protein signaling cascade while tracking multiple components simultaneously. Specifically, we quantitatively measure signaling through Gαi-coupled GPCRs in HEK293 and MIN6 cell lines.