Work

Engineered interfaces in Rac1 and Cdc42 biosensors enhance sensitivity and reduce cell perturbation

Year: 2026

Type: article

Abstract: Fluorescent biosensors are a valuable means to report the spatiotemporal dynamics of protein activities in live cells and animals. However, biosensors affect the activities they are reporting. This can be ameliorated by increasing sensitivity, using lower biosensor concentrations, or by choosing designs that minimize undesirable interactions. For biosensors in which fluorescent components interact to produce Forster Resonance Energy Transfer (FRET), perturbation is often due to interaction of biosensor components with nonfluorescent, endogenous proteins, rather than productive interactions that lead to FRET. Here, we engineer the interface between biosensor components using charge swap and "knob into hole" mutations to reduce all but desired interactions. Novel biosensors for Rac1 and Cdc42 showed reduced interactions with endogenous GTPases and effectors, normal activation by guanine nucleotide exchange factors (GEFs), and correctly reproduced previous reports of GTPase activation dynamics. Assaying concentration-dependent effects on cell motility showed substantially reduced perturbation of normal cell behavior. Computational models indicated that minimal perturbation could be achieved over a broader range of concentrations using the new "orthogonal" biosensors.

Source: Molecular Biology of the Cell

DOI: 10.1091/mbc.e25-10-0494