Single-Component Optogenetic Tools for Inducible RhoA GTPase Signaling
Erin E Berlew1, Ivan A Kuznetsov1, Keisuke Yamada2
1Department of Bioengineering, University of Pennsylvania, 210 South 33 rd Street, Philadelphia, PA, 19104, USA.
Advanced Biology
|July 21, 2021
Summary
New optogenetic tools precisely control RhoA GTPase, a key regulator of cell structure. This light-activated system enables rapid manipulation of the actin cytoskeleton, influencing cell shape and gene expression.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Biology
Background:
- RhoA GTPase is a critical regulator of the actin cytoskeleton and actomyosin contractility.
- Controlling RhoA signaling with spatiotemporal precision is essential for understanding cellular processes.
Purpose of the Study:
- To develop novel optogenetic tools for precise spatiotemporal control of RhoA GTPase activity.
- To investigate the effects of light-induced RhoA signaling on cytoskeletal organization, cell-cell junctions, and mechanotransduction.
Main Methods:
- Engineering RhoA GTPase or its activator ARHGEF11 fused to the photoreceptor BcLOV4.
- Utilizing light-regulated protein-lipid electrostatic interactions for dynamic plasma membrane recruitment.
- Applying patterned blue light stimulation to induce localized cytoskeletal changes.
- Assessing adherens junction separation, cytoskeletal morphology, and YAP nuclear localization via microscopy and transcriptional activity assays.
Main Results:
- A single pulse of blue light induced potent contractile signaling and adherens junction separation.
- Light-induced cytoskeletal changes were dependent on the alignment of stimulation with cell polarization.
- Activated RhoA signaling rapidly drove YAP nuclear localization and transcriptional activity, indicating mechanotransduction.
- The developed optogenetic tools enabled precise control over RhoA signaling without requiring additional protein binding partners.
Conclusions:
- Novel optogenetic tools provide unprecedented spatiotemporal control over RhoA signaling.
- Light-induced RhoA activation can rapidly alter cell morphology, cell-cell adhesion, and downstream signaling pathways.
- These tools facilitate advanced studies into the multifaceted roles of RhoA in cell migration, morphogenesis, and cell cycle regulation.
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