Towards characterizing the biophysical landscape of immune cells with a novel modular toolbox.*

Cellular membranes comprise hundreds of unique lipid species whose interactions result in a variety of membrane properties such as lipid packing, membrane thickness, and membrane fluidity. These biophysical properties, in combination with preferential sorting of lipids, regulate membrane-associated proteins which represent approximately 60% of drug targets and are encoded by more than 30% of the human proteome. Investigating the influence of local lipid environments – functionally termed the paralipidome – may provide revolutionary insight into fundamental biological mechanisms. For example, the Linker for Activation of T-Cells (LAT), a critical regulator of immune cell activation, is thought to be strongly modulated by its lipid environment. We present a modular HaloTag-based toolbox that integrates environment-sensitive fluorescent probes, previously applied to measure bulk membrane properties, to directly measure the biophysical properties of the paralipidome near specific proteins. We report successful implementation and validation of this approach and highlight ongoing progress in applying the toolbox to characterize the local lipid environment of T-cells before and after immune cell activation.