GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System

Abstract

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.

Light-gated Integrator for Highlighting Kinase Activity in Living Cells

“…As expected, cells treated with F/I showed a sustained increase in the H2B- EGFP signal, whereas Iso stimulation only induced a transient increase in H2B-EGFP fluorescence. Tracing the accumulation of fluorescence during each time window reflects the dynamic changes in PKA activity induced under the different stimulation conditions, which are largely consistent with past results obtained using real-time PKA biosensors 9, 10 . These data reveal that the two components of the KINACT system can reversibly combine and separate in response to changes in kinase activity, thus providing an accurate readout of kinase activity levels during the corresponding recording window.…”

Sensitive Fluorescent Biosensor Reveals Differential Subcellular Regulation of PKC

“…As shown in Figure 3a, stimulating Cos7 cells expressing ER-ExRai-CKAR2 or Lyso-ExRai-CKAR2 with UTP (100 μM) induced clear elevations in PKC activity at each location, with a 21% ± 2% excitation ratio increase (ΔR/R 0 , n = 36 cells) from ER-ExRai-CKAR2 and a 40% ± 2% excitation ratio increase (ΔR/R 0 , n = 36 cells) from Lyso-ExRai-CKAR2. We next assessed the dynamics of agonist-induced PKC activity at these two locations by quantifying the fraction of sensor response remaining at 15 min after stimulation versus the maximum response (i.e., sustained activity metric at 15 min, or SAM15) 41,42 . Interestingly, both ER and lysosomal PKC activities were sustained after UTP stimulation, with a SAM15 of 0.65 ± 0.02 (n = 36 cells) at the ER and a SAM15 of 0.92 ± 0.05 (n = 36 cells) at the lysosome (Figure 3a).…”

Fluorescent biosensor imaging meets deterministic mathematical modelling: quantitative investigation of signalling compartmentalization

“…New experimental techniques enable enhanced throughput for quantitative, single‐cell dynamic measurements (Greenwald et al., 2023) as well as investigations of many different signalling components simultaneously, such as improvements in biosensor multiplexing strategies in single cells (Mehta et al., 2018) or biosensor barcoding schemes in populations of cells (Yang et al., 2021). With increased information, computational models can serve as a quantitative framework to integrate datasets and enable investigation of complex network behaviour.…”