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Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)
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Imaging CREB activation in living cells.

Michael W Friedrich1, Gayane Aramuni, Marco Mank

  • 1Max-Planck-Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany.

The Journal of Biological Chemistry
|May 21, 2010
PubMed
Summary
This summary is machine-generated.

Researchers developed a new indicator to visualize calcium and cAMP-responsive element-binding protein (CREB) activation in single cells. This tool reveals AKAP79/150

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Area of Science:

  • Molecular Biology
  • Cellular Signaling
  • Neuroscience

Background:

  • Calcium and cAMP-responsive element-binding protein (CREB) are crucial transcription factors linking cellular activity to gene expression.
  • Dynamic imaging of CREB activation in single cells is limited by current biochemical technologies.
  • Understanding CREB regulation is vital for deciphering activity-dependent gene expression.

Purpose of the Study:

  • To develop a novel genetically encoded indicator for dynamic imaging of CREB activation.
  • To investigate the role of AKAP79/150 in regulating CREB activation pathways.
  • To assess the contribution of protein kinase A anchoring and L-type channels in CREB regulation.

Main Methods:

  • Developed a signal-optimized genetically encoded indicator using fluorescence resonance energy transfer (FRET) between green fluorescent protein mutants.
  • Named the indicator ICAP (indicator of CREB activation due to phosphorylation).
  • Utilized ICAP to image CREB activation in living cells, including experiments involving disruption of AKAP79/150-mediated protein kinase A anchoring and AKAP150 knock-down.

Main Results:

  • The developed ICAP indicator enables dynamic and reversible imaging of CREB activation in living cells.
  • Disruption of AKAP79/150-mediated protein kinase A anchoring significantly reduces protein kinase A's ability to activate CREB.
  • AKAP150 knock-down also dramatically reduces protein kinase A-mediated CREB activation, indicating a critical role for AKAP150.
  • AKAP79/150 regulation of CREB via L-type channels appears to have a minor role.

Conclusions:

  • The novel ICAP indicator provides a powerful tool for real-time visualization of CREB phosphorylation at serine 133.
  • AKAP79/150 plays a significant role in regulating CREB activation, primarily through its scaffolding function for protein kinase A.
  • ICAP has the potential to advance the study of molecular components and cell-type specificity in activity-dependent gene expression.