This $186,250 Project Grant award from the National Cancer Institute's Cancer Biology Research program (CFDA 93.396) is funding the development and characterization of novel fluorescent probes to measure the nano-scale membrane environments surrounding membrane proteins in living cells. The primary awardee, the University of Virginia, intends to employ a combined simulation-experiment approach to calibrate various membrane-sensitive fluorescent sensors and determine their spatial range and sensitivity to specific membrane structural parameters. The university will then demonstrate proof-of-concept applications of these probes to measure how membrane protein "paralipidomes" - the local lipid environments - evolve through the secretory pathway and how lipid asymmetry across cell membranes creates biophysical gradients around membrane proteins. Completing these aims will provide a validated toolbox for probing how membrane proteins assemble and regulate their local lipid environments, which is critical for understanding fundamental membrane protein biology.