Monitoring Cellular Energy Balance in Single Cells Using Fluorescent Biosensors for AMPK

Abstract

5'-Adenosine monophosphate-activated protein kinase (AMPK) senses cellular metabolic status and reflects the balance between ATP production and ATP usage. This balance varies from cell to cell and changes over time, creating a need for methods that can capture cellular heterogeneity and temporal dynamics. Fluorescent biosensors for AMPK activity offer a unique approach to measure metabolic status nondestructively in single cells in real time. In this chapter, we provide a brief rationale for using live-cell biosensors to measure AMPK activity, survey the current AMPK biosensors, and discuss considerations for using this approach. We provide methodology for introducing AMPK biosensors into a cell line of choice, setting up experiments for live-cell fluorescent microscopy of AMPK activity, and calibrating the biosensors using immunoblot data.

Keywords: AMPKAR; Biosensors; Fluorescent protein reporters; Forster resonance energy transfer (FRET); Live-cell microscopy; Metabolic signaling; Single cell.

Figure 1:

Example of AMPKAR calibration with immunoblot data. (A) Example mean live-cell AMPKAR data across conditions used to calibrate the sensor. (B) Example phosphate binding immunoblot performed on cell lysates from calibration experiments. (C) Scatter plot with fit line showing AMPKAR FRET activity from Figure 1A at hour 3 for each condition versus relative fraction AMPKAR biosensor phosphorylated to unphosphorylated shown in Figure 1B. Line is fit to data and used to normalize AMPKAR data scaling 0 to 1.