High-throughput screening to identify modulators of membrane protein raft affinity in giant plasma membrane vesicles

Lipid rafts remain an active area of membrane biophysics research. Due to their diffraction-limited size and transient nature direct studies of rafts and their resident proteins in cells remains intractable. The lack of tools to manipulate raft-resident proteins has also stymied our ability to study raft function and raft partitioning of proteins. Giant plasma membrane vesicles (GPMVs), which spontaneously separate into ordered (raft) and disordered (non-raft) phases, are a practical a tool for studying rafts in biological membranes. We recently developed a high-throughput screening pipeline combining high-content imaging with a custom image-analysis software package, VesA, to identify small molecule modulators of phase separation in GPMVs. Using this approach, we are conducting an on-going screen 20,000 of small molecules from the Vanderbilt University Discover collection to find compounds that alter the phase partitioning of the peripheral myelin protein 22 (PMP22). PMP22, a major component of myelin in Schwann cells of the peripheral nervous system, shows a high affinity for ordered phases of GPMVs. Disease causing mutations in PMP22 exhibit both varying degrees of misfolding and decreased raft partitioning which strongly correlate to disease severity. We hypothesize that PMP22 fold stability is necessary for both raft partitioning and trafficking to the plasma membrane. From the screen, we are identifying hits that we sort into different categories based on their effects on (1) PMP22 raft partitioning (2) GPMV phase composition and (3) raft partitioning of other proteins. Identification of small molecules that non-specifically alter raft affinity will be useful tools for dissecting how and why proteins prefer rafts. The identification of small molecules that selectively bind to PMP22 and alter its phase partitioning will be crucial to investigating the relationship between PMP22 structure, raft affinity, and trafficking.