Cell-based optimisation and characterisation of genetically encoded, location-based biosensors for Cdc42 or Rac activity

Rac and Cdc42 are Rho GTPases which regulate the formation of lamellipoda and filopodia and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for either Rac or Cdc42. We compared their (i) ability to bind the constitutively active Rho GTPases, (ii) specificity for Rac and Cdc42 and (iii) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1 we found a sensor candidate with low relocation efficiency. For Cdc42 we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. The characterization and optimization of relocation sensors will broaden their application and acceptance. ### Competing Interest Statement The authors have declared no competing interest.