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Related Experiment Video

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Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis
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Akt signaling dynamics in individual cells.

Sean M Gross1, Peter Rotwein2

  • 1Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA.

Journal of Cell Science
|June 5, 2015
PubMed
Summary

Researchers developed a novel fluorescent reporter to track Akt kinase activity in single cells. This tool reveals variable Akt signaling at the single-cell level despite stable population responses, advancing our understanding of Akt biology.

Keywords:
AktIGF-ILive-cell imagingSignaling dynamicsSignaling pathways

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Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • The protein kinase Akt is a crucial intracellular mediator involved in numerous biological processes.
  • Limited understanding of Akt signaling dynamics hinders comprehensive knowledge of its regulatory roles.
  • Investigating Akt activity at the single-cell level is essential for elucidating its complex biological functions.

Purpose of the Study:

  • To develop and validate a novel fluorescent reporter for real-time, single-cell assessment of Akt kinase activity.
  • To investigate the dynamics and variability of Akt signaling in response to stimulation.
  • To provide a versatile tool for studying Akt-mediated biological processes across different experimental platforms.

Main Methods:

  • Construction of a lentiviral delivery system encoding a fluorescent reporter molecule.
  • The reporter is a fusion protein comprising a modified FoxO1 transcription factor and a green fluorescent protein (clover).
  • Tracking of reporter translocation from the nucleus to the cytoplasm in response to Akt stimulation.

Main Results:

  • The developed sensor exhibits a long half-life and high fluorescence intensity, enabling robust, long-term tracking of Akt activity.
  • Akt stimulation by IGF-I elicits stable, reproducible analog responses at the population level.
  • Significant variability in Akt signaling outcomes was observed at the single-cell level.

Conclusions:

  • The novel fluorescent reporter provides a simple, dynamic, and reliable measure of Akt activity at the single-cell level.
  • This tool is compatible with various cell types and experimental setups, facilitating broader research applications.
  • The findings highlight the heterogeneity of single-cell Akt signaling, offering new insights into Akt's regulatory mechanisms.