A Far-Red FRET biosensor for AMPK enables multiplexed imaging of single-cell bioenergetic homeostasis

Nicholaus L. DeCuzzi1*, Marion Hardy1*, Markhus B. Cabel1, Jason Hu1, Christina Abbate1, Michael Pargett1, and John G. Albeck1♱.

Affiliations: 1 Department of Molecular and Cellular Biology, University of California, Davis. *These Authors contributed equally. ♱To whom correspondence should be addressed: jgalbeck@ucdavis.edu

Abstract

Metabolic homeostasis has been studied primarily at the tissue and organism level, identifying molecular control mechanisms such as the energy charge-sensing kinase AMPK. Feedback loops involving AMPK and other regulators align cellular ATP generation and consumption, determining overall energetic balance. Recent work has demonstrated surprising oscillatory dynamics in AMPK activity, suggesting unknown mechanisms in single-cell homeostatic behavior. Probing intracellular feedback requires simultaneous observation of multiple energetic parameters, but such experiments are precluded by the shared wavelength band occupied by most metabolic biosensors. We have overcome this obstacle by constructing a red-shifted FRET-based AMPK activity biosensor, RAMPKAR2, that is comparable to existing FRET-based AMPK activity biosensors. Multiplexed imaging of RAMPKAR2 with Perceval-HR, which detects ATP/ADP ratio, confirmed that the kinetics of AMPK activity and ATP/ADP ratio are tightly coupled, with a lag of less than 6 minutes at the single-cell level. Coupling of RAMPKAR with HYlight, which detects the glycolytic intermediate fructose 1,6-bisphosphate (FBP), revealed that glycolytic activity co-oscillates with AMPK, and that these oscillations are suppressed by sustained AMPK activity. Together these data support a model in which temporally offset increases in glycolytic ATP supply and AMPK deactivation contribute to single-cell oscillations.

Content of the repository

Replicates

• Labeled as yyyy-mm-dd_CellLine_Modification_Sensor
• Code for general figure production
• Timeseries figures : Averages and single cell traces

Plasmid maps

• In snapgene-compatible formats

Code for figure generation

• data/
• figures/
• stats/
• Scripts per figure panels

Note

If you are trying to replicate the results and need access to some dependencies, please contact jgalbeck@ucdavis.edu, so that we may provide those scripts.