(837.7) GPCR signaling measurement and drug profiling with an automated live-cell microscopy system

Poster Board Number: B66

Clara Posner (University of California), Eric Greenwald (Pfizer), Ananya Bharath (University of California), Anne Lyons (University of California), Cristina Salmerón (University of California), Jane Smitham (University of California), Paul Insel (University of California), Jin Zhang (University of California)

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.

This work was supported by the R35 CA197622 and R01 DK073368 to Jin Zhang.