Quantifying the Impact of Signaling Molecule Concentration on the Intercommunication of Sensory Protein Vesicles

Bottom-up synthesis of synthetic cells holds the potential to enhance our comprehension of foundational biological principles and unlock biomedical applications, such as smart drug delivery systems. Globular protein vesicles (GPVs), formed through self-assembly using recombinant fusion proteins, stand as a promising platform for this purpose, due to their tunable properties and biocompatible production method. Herein, our work aims to develop and quantify sensory functions within the GPV platform, an important step towards constructing GPV-based synthetic cells. The designed sensory GPV system relies on the established protein binding interaction involving rapamycin, FKBP, and FRB. FKBP and FRB were incorporated into the GPV membranes, which were then exposed to specific concentrations of rapamycin, acting as a signaling molecule. A sensory response of FKBP- and FRB-decorated GPVs to rapamycin was indicated by the aggregation of the vesicles. It was found that the extent of intercommunication among sensory protein vesicles is impacted by the concentration of the signaling molecule. Quantitative analysis of fluorescent micrographs using ImageJ/FIJI with computing a coefficient of colocalization demonstrated that the variation in sensing extent across different working concentrations of the signaling molecule. Additionally, a range of optimal signaling molecule concentrations for maximal sensing, as well as upper and lower limits to sustain vesicle stability while inducing sensing. The same quantitative approach verified that the introduction of FKBP and FRB proteins to the GPV membrane is responsible for the sensory response to the presence of rapamycin. This work would provide a fundamental basis for integrating sensory protein functions into synthetic cells. This analysis method can also facilitate comparisons of intercommunication between vesicles with diverse characteristics.