A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FAL-COscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.
Time‐lapse microscopy combined with genetically encoded fluorescent biosensors is a powerful tool for quantitatively measuring live cell signaling dynamics with high spatial and temporal resolution in single cells, providing rich signaling information beyond population‐based biochemical assays and plate reading measurements. One major limitation, however, is the low experimental throughput. To overcome this limitation, we created a highly customizable MATLAB‐based platform called FALCOscope (Flexible Automated Liquid‐handling Combined Microscope) that coordinates between an OpenTrons liquid handler and a fluorescent microscope to automate drug stimulations, fluorescence imaging and single cell analysis. We demonstrated the feasibility of our approach by quantifying GPCR‐stimulated Protein Kinase A (PKA) activity dynamics and by measuring cAMP dynamics stimulated by a panel of 14 GPCR agonists and antagonists. We then characterized the cAMP dynamics induced by GPR68, a proton sensing GPCR, in response to extracellular pH changes in the presence of its positive allosteric modulator ogerin. We found that GPR68 induced cAMP responses are more transient for acidic pH’s than neutral pH’s, suggesting receptor desensitization at acidic pHs. We further identified that ogerin, in addition to enhancing the cAMP response mostly strongly for pH 7.0, also sustains the cAMP response for acidic pH’s, demonstrating the novel findings captured by live‐cell dynamics. In addition, we show that 100 µM ogerin also has off‐target effects on cAMP/PKA signaling, likely as a phosphodiesterase inhibitor. This FALCOscope system enables single‐cell dynamic measurement in high throughput, providing a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells as well as drug profiling and screening.
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