Spatial Cumulant Analysis to Study D2-Like Dopamine Receptor Dynamics on Plasma Membrane

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Fluorescence fluctuation spectroscopy (FFS) measures physical properties of fluorescent samples by analyzing the statistical nature of signals measured by photodetectors. In biological contexts, the most frequently measured parameters include concentration, molecular brightness, and motion characteristics (i.e. diffusion coefficients). Molecular brightness holds salience because it can be used to determine the stoichiometry of homomeric and heteromeric interactions between fluorescent species. We use spatial cumulant analysis (SpCA), an adaptation of cumulant analysis of point measurements, to determine molecular brightness from images recorded using two-photon laser scanning microscopy. Spatial cumulant analysis combines the advantages of imaging and single point cumulant analysis. Imaging provides decreased photobleaching, the ability to measure fluctuations from a larger set of molecules, and spatial resolution of the measured parameters. Cumulants are favorable in FFS because they are additive for multiple molecular species and provide computational ease. Accurate determination of stoichiometry from molecular brightness can be difficult for several reasons including non-ideal photodetection, incomplete chromophore maturation, photobleaching, and resonance energy transfer. Our approach accounts for these complications. D2-like dopamine receptors (D2Rs) are cell surface receptors that couple to trimeric G-protein complexes. Activation of D2Rs catalyzes the dissociation of the G-protein complex into Gα and Gβγ subunits. Gα and Gβγ subunits mediate the action of D2Rs by interaction with downstream effectors including adenylyl cyclase, inwardly rectifying K+ channels, and voltage-gated Ca2+ channels. Physiologically, D2R activation inhibits cAMP production and Ca2+ influx while activating hyperpolarizing K+ currents. While it is known that D2Rs, G-proteins, and their effectors interact to produce these physiological effects, the details of these interactions are difficult to probe experimentally in live cells. We show how multicolor spatial cumulant analysis of labeled proteins of interest can accurately measure the stoichiometry of receptors, G-proteins, and downstream signaling targets.