AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But how AMPK activity is regulated at different subcellular locations for precise spatiotemporal control over metabolism is unclear. Here we present a sensitive, single-fluorophore AMPK activity reporter (ExRai AMPKAR), which reveals distinct kinetic profiles of AMPK activity at the mitochondria, lysosome, and cytoplasm. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) results in slower AMPK activity at lysosomes but does not affect the response amplitude at lysosomes or mitochondria, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further identify a mechanism for AMPK activity in the nucleus, which results from cytoplasmic to nuclear shuttling of AMPK. Thus, ExRai AMPKAR enables illumination of the complex subcellular regulation of AMPK signaling.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.