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Published on: November 2, 2018
Activation of NF-κB Signaling by Optogenetic Clustering of IKKα and β
Alexandra Anna Maria Fischer1,2,3,4, Markus Michael Kramer1,2, Miguel Baños4,5
1Signalling Research Centers BIOSS and CIBSS, University of Freiburg, Schänzlestr. 18, 79104, Freiburg, Germany.
Abstract:
Molecular optogenetics allows the control of molecular signaling pathways in response to light. This enables the analysis of the kinetics of signal activation and propagation in a spatially and temporally resolved manner. A key strategy for such control is the light-inducible clustering of signaling molecules, which leads to their activation and subsequent downstream signaling. In this work, an optogenetic approach is developed for inducing graded clustering of different proteins that are fused to eGFP, a widely used protein tag. To this aim, an eGFP-specific nanobody is fused to Cryptochrome 2 variants engineered for different orders of cluster formation. This is exemplified by clustering eGFP-IKKα and eGFP-IKKβ, thereby achieving potent and reversible activation of NF-κB signaling. It is demonstrated that this approach can activate downstream signaling via the endogenous NF-κB pathway and is thereby capable of activating both an NF-κB-responsive reporter construct as well as endogenous NF-κB-responsive target genes as analyzed by RNA sequencing. The generic design of this system is likely transferable to other signaling pathways to analyze the kinetics of signal activation and propagation.
Insights
Researchers developed a novel optogenetic tool for light-controlled protein clustering. This method enables precise control over molecular signaling pathways, like NF-κB, for detailed kinetic analysis.
Area of Science:
- Molecular Biology
- Optogenetics
- Cell Signaling
Background:
- Optogenetics offers light-based control over molecular signaling pathways.
- Light-inducible protein clustering is a key strategy for activating signaling cascades.
- Analyzing signaling kinetics requires precise spatial and temporal resolution.
Purpose of the Study:
- To develop a novel optogenetic system for inducing graded protein clustering.
- To demonstrate the system's ability to control and analyze signaling pathway activation.
- To validate the system's applicability to endogenous signaling pathways.
Main Methods:
- Engineered Cryptochrome 2 variants fused to an eGFP-specific nanobody.
- Fusion of eGFP-tagged proteins (e.g., IKKα, IKKβ) to the nanobody system.
- Activation of NF-κB signaling pathway via light-induced clustering.
- Analysis of downstream effects using reporter constructs and RNA sequencing.
Main Results:
- Achieved potent and reversible activation of NF-κB signaling through graded protein clustering.
- Demonstrated activation of endogenous NF-κB-responsive reporter constructs.
- Confirmed activation of endogenous NF-κB target genes via RNA sequencing.
- Showcased the system's capacity for spatially and temporally resolved signaling analysis.
Conclusions:
- The developed optogenetic system provides a versatile platform for controlling protein clustering.
- This approach enables detailed kinetic analysis of signal activation and propagation.
- The system's generic design is transferable to various signaling pathways for research applications.
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