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A new single chain, genetically encoded biosensor for RhoB GTPase based on FRET, useful for live-cell imaging.

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Researchers developed a new biosensor to visualize RhoB GTPase activity in living cells. This tool reveals coordinated RhoB and TC10 activities during cell migration, advancing our understanding of membrane trafficking and signaling.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • RhoB GTPase plays a crucial role in membrane trafficking and cytoskeletal regulation.
  • Its function is closely tied to subcellular localization and endosomal/plasma membrane association.
  • Existing tools lack the ability to monitor RhoB activity in real-time and space.

Purpose of the Study:

  • To develop and validate a genetically encoded Förster resonance energy transfer (FRET) biosensor for RhoB.
  • To enable direct visualization of RhoB activity in living cells.
  • To investigate the spatiotemporal coordination of RhoB and TC10 activities during cell migration.

Main Methods:

  • Development of a single-chain, genetically encoded FRET biosensor for RhoB.
  • Validation of the biosensor in living cells, preserving native membrane-targeting determinants.
  • Multiplex live-cell imaging combining RhoB and TC10 FRET biosensors.
  • Quantitative morphodynamic and cross-correlation analyses.

Main Results:

  • The developed RhoB biosensor has a large dynamic range and visualizes heterogeneous RhoB activity during cell migration.
  • Multiplex imaging revealed coordinated yet antagonistic spatiotemporal patterns of RhoB and TC10 activities at the cell leading edge.
  • Perturbation of TC10 regulation altered the spatial coupling between RhoB and TC10.

Conclusions:

  • A robust FRET biosensor for RhoB activity has been established.
  • A multiplex imaging framework allows for studying the coordination of trafficking and signaling pathways.
  • This work provides new insights into the regulation of cell migration by Rho GTPases.