Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

45-POS Board 12 HIGH-THROUGHPUT SCREENING TO IDENTIFY MODULATORS OF RAFT FORMATION AND MEMBRANE PROTEIN RAFT AFFINITY IN GIANT PLASMA MEMBRANE VESICLES Katherine M Stefanski 1 ; Hui Huang 1 ; Joshua A Bauer 1 ; Anne K Kenworthy 2 ; Charles R Sanders 1 ; 1 Vanderbilt University, Biochemistry, Nashville, TN, USA 2 University of Virginia, Charlottesville, VA, USA Lipid rafts remain an intriguing and 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. Giant plasma membrane vesicles (GPMVs), which spontaneously separate into ordered (raft) and disordered (non-raft) phases, have emerged as practical a tool for studying rafts in biological membranes. The inability to selectively manipulate rafts and raft- resident proteins has also stymied our ability to study raft formation and the partitioning of proteins into them. We recently developed a high-throughput screening pipeline to identify small molecule modulators of phase separation in GPMVs. Combining high-content imaging with a custom image-analysis software package, VesA, we successfully identified compounds which robustly increase and decrease phase separation in GPMVs. From this proof-of-concept study, we next sought 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 mutations in PMP22 exhibit both varying degrees of misfolding and decreased raft partitioning. The identification of small molecules that selectively bind to PMP22 and alter its phase partitioning will be crucial to investigate how and why PMP22 partitions into rafts. We conducted a preliminary screen of PMP22 partitioning with a library of 1100 FDA approved drugs. A number of non-specific partitioning modulators were identified. We then conducted a screen of 20,000 small molecules from the Vanderbilt University Discover collection. Hits from these screens will be invaluable to our efforts to understand the relationship between PMP22 structure and raft affinity.

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