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

Nicholaus DeCuzzi1, Nont Kosaisawe1, Michael Pargett1

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Summary

Live-cell fluorescent biosensors enable real-time measurement of 5'-Adenosine monophosphate-activated protein kinase (AMPK) activity. This approach captures cellular metabolic heterogeneity and temporal dynamics, offering a non-destructive method for studying cellular energy balance.

Keywords:
AMPKARBiosensorsFluorescent protein reportersForster resonance energy transfer (FRET)Live-cell microscopyMetabolic signalingSingle cell

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Metabolic Engineering

Background:

  • 5 -Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, sensing the ATP/AMP ratio.
  • Cellular metabolic status exhibits significant heterogeneity and dynamic changes over time.
  • Existing methods for assessing AMPK activity often lack the spatiotemporal resolution required to capture these dynamics in single cells.

Purpose of the Study:

  • To introduce and validate the use of live-cell fluorescent biosensors for real-time, non-destructive measurement of AMPK activity.
  • To provide a comprehensive guide for implementing and utilizing AMPK biosensors in cellular studies.
  • To address the need for methods capturing cellular heterogeneity and temporal dynamics in AMPK activity.

Main Methods:

  • Development and application of fluorescent biosensors for detecting AMPK activity.
  • Introduction of biosensors into chosen cell lines.
  • Live-cell fluorescent microscopy for real-time monitoring of AMPK activity.
  • Calibration of biosensor data using immunoblotting.

Main Results:

  • Fluorescent biosensors provide a powerful tool for visualizing AMPK activity in single cells.
  • The methodology allows for the capture of dynamic changes in cellular metabolic status.
  • Biosensor calibration ensures quantitative and reliable measurements of AMPK activity.

Conclusions:

  • Live-cell fluorescent biosensors are a valuable and versatile approach for studying AMPK activity.
  • This technique facilitates the investigation of cellular metabolic heterogeneity and real-time responses.
  • The provided methodology enables researchers to effectively implement and utilize AMPK biosensors in their studies.