posted on 2023-01-05, 17:14authored byEric Greenwald, Clara Posner, Ananya Bharath, Anne Lyons, Cristina Salmerón, Krishna Sriram, Shu Z. Wiley, Paul A. Insel, Jin Zhang
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 (FALCOscope)
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.