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Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac

Eike K Mahlandt1, Gabriel Kreider-Letterman2, Anna O Chertkova1

  • 1Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

Journal of Cell Science
|May 25, 2023
PubMed
Summary
This summary is machine-generated.

Researchers developed and optimized relocation biosensors for Rac and Cdc42 Rho GTPases, crucial for cell migration. These improved sensors enhance the study of cell signaling and enable detection of local Rho GTPase activity.

Keywords:
AffinityBiosensorCdc42Fluorescent proteinMultiplex imagingRho GTPase

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Rho GTPases, including Rac and Cdc42, are key regulators of actin cytoskeleton dynamics, controlling cell migration through lamellipodia and filopodia formation.
  • Existing relocation-based biosensors for Rac and Cdc42 lack thorough characterization regarding specificity and affinity, limiting their application.
  • Understanding Rho GTPase activity is vital for deciphering cellular processes like migration and invasion.

Purpose of the Study:

  • To identify and characterize novel relocation sensor candidates for Rac and Cdc42.
  • To compare the binding ability, specificity, and relocation efficiency of these sensor candidates.
  • To optimize sensor performance for enhanced application in studying Rho GTPase activity.

Main Methods:

  • Screening and identification of potential Rac and Cdc42 relocation sensor candidates.
  • Comparative analysis of sensor candidates' binding affinity to constitutively active Rho GTPases.
  • Evaluation of sensor specificity for Rac and Cdc42 in cell-based assays.
  • Optimization of relocation efficiency using a multi-domain approach.
  • Testing of various fluorescent proteins and HaloTag for optimal recruitment efficiency in multiplexing experiments.

Main Results:

  • Identified sensor candidates for both Rac and Cdc42, with varying degrees of relocation efficiency and specificity.
  • A sensor candidate for Rac1 exhibited low relocation efficiency.
  • Several sensors for Cdc42 demonstrated sufficient relocation efficiency and specificity.
  • Optimized sensors successfully detected local endogenous Cdc42 activity at assembling invadopodia.
  • Characterized the influence of different fluorescent proteins and HaloTag on sensor recruitment efficiency.

Conclusions:

  • Developed and optimized relocation biosensors for Rac and Cdc42, improving their specificity and efficiency.
  • The optimized Cdc42 sensors are suitable for detecting localized Rho GTPase activity, as demonstrated at invadopodia.
  • This work provides a valuable toolkit for broader application and acceptance of Rho GTPase relocation sensors in cell biology research.
  • Findings facilitate multiplexing experiments by identifying optimal components for Rho location sensors.