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Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE
Julia C Hardy1, Sohum Mehta2, Jin Zhang3,4,5
1Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 14, 2022
Summary
This study introduces FluoSTEP-ICUE, a novel method using CRISPR-Cas9 to visualize cyclic adenosine monophosphate (cAMP) signaling dynamics in specific cellular locations. This technique allows for precise measurement of cAMP levels within endogenous signaling compartments.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biochemistry
Background:
- Cyclic adenosine monophosphate (cAMP) is a critical second messenger regulating numerous cellular functions.
- Precise spatiotemporal control of cAMP signaling, achieved through compartmentalization in subcellular microdomains, is vital for cellular specificity.
- Existing methods often rely on overexpressing biosensor-tagged proteins, which can disrupt native cellular processes.
Purpose of the Study:
- To develop and present a novel protocol for directly visualizing signaling dynamics within cAMP microdomains.
- To enable the measurement of cAMP levels at endogenous protein locations within specific cellular compartments.
Main Methods:
- Development of FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE), a FRET-based cAMP biosensor.
- Utilizing spontaneously complementing split GFP for biosensor assembly.
- Employing CRISPR-Cas9 genome editing to target the biosensor to endogenously expressed proteins of interest.
Main Results:
- Successfully localized a FRET-based cAMP biosensor to endogenous proteins using CRISPR-Cas9.
- Demonstrated the ability to measure cAMP levels within endogenous signaling compartments.
- Provided a method for direct visualization of cAMP microdomain signaling dynamics.
Conclusions:
- FluoSTEP-ICUE offers a powerful tool for studying the spatiotemporal regulation of cAMP signaling.
- This approach overcomes limitations of traditional methods by avoiding protein overexpression.
- Enables high-resolution analysis of cAMP dynamics in native cellular contexts.
