Quantitative Hydrophobicity Sensor to Probe the Dynamics of Biomolecular Condensates

PROJECT SUMMARY Many important cellular processes are regulated through the formation and dissolution of the biomolecular condensates via liquid-liquid phase separation (LLPS). LLPS enriches specific factors in the biomolecular con- densates while excluding others, thereby creating a unique environment that either promotes or restricts certain biochemical reactions. To investigate how the dynamic process of LLPS and the reverse process that results in the dissolution of biomolecular condensates, biosensors capable of survey the biophysical properties of conden- sates as they form and dissolve within cells are highly desirable. The stability of a biomolecular condensate depends on electrostatic forces as well as hydrophobic interactions between the molecules residing in the con- densate. Currently there are no known biosensors for real-time monitoring of environmental hydrophobicity in living cells, limiting our understanding of how hydrophobicity changes over the lifetime of biomolecular conden- sates. Here we propose to develop a genetically encoded hydrophobicity biosensor, consisting of a pair of fluo- rescent proteins that can undergo Förster Resonance Energy Transfer (FRET). This hydrophobicity sensor will report FRET efficiency as the readout of hydrophobicity value. As proof of concept, we will create a recombinant protein in which the fluorescent profiting pair is fused with paxillin, an important protein in neurite growth, migra- tion of neuron and microglial cell, as well as endocytosis in cells of the neural system. We plan to first establish a calibration curve by which hydrophobicity can be quantified. Then we plan to demonstrate that this hydropho- bicity sensor can be used intracellularly to monitor the hydrophobicity changes in biomolecular condensates to which paxillin partitions. If successful, our design principle can be readily applied to measure hydrophobicity of condensates containing other molecules.