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

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.