Abstract
Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.
Highlights
Membrane rafts represent an extensively studied yet persistently enigmatic example of how membrane organization can modulate cellular function.[1]
Effects of small molecules or other treatments on membrane phase behavior are evaluated by cdoitmiopna.1r1in,1g6,12T,1m9,i2sc2−m24eaTshuirsemapepnrtosacohbtiasinneodt under each practical for conhighthroughput screening applications, which typically are performed at a single temperature
We have provided a proof of concept for a highthroughput method to discover chemical modulators of membrane rafts
Summary
Membrane rafts represent an extensively studied yet persistently enigmatic example of how membrane organization can modulate cellular function.[1] Typically defined as nanoscopic cholesterol-enriched domains that share properties with liquid ordered domains in vitro, rafts coexist with disordered domains in cell membranes and regulate numerous cellular functions by controlling the interaction partners and proximal membrane environment of associated proteins.[1] Consistent with their extensive biological roles, rafts have been implicated in a variety of normal physiological processes as well as pathological conditions.[1−6] Raft-dependent processes are typically identified and manipulated by altering membrane lipid composition, most commonly via cholesterol depletion.[1] such approaches can have significant pleiotropic effects and nonspecifically affect multiple lipid-dependent pathways.[7,8] Another possible, though not yet widely explored, approach to modulate rafts in biological membranes is through the use of small molecules.[2−5,9] For example, specific therapeutics that can modulate microdomains are being actively considered for development.[5] Besides targeting specific lipids that alter receptor signaling, certain drugs can modulate general properties of membrane rafts.[10,11] in cancer cell lines, changes in membrane heterogeneity are correlated with resistance to chemotherapeutics, and altering membrane heterogeneity can modulate cellular responses to chemotherapeutic drugs.[12] chemically based strategies could potentially provide new experimental tools to manipulate rafts in vitro and even eventually establish new therapeutics for human diseases linked to raft biology. Tmisc is sensitive to the effect of small molecules and bioactive compounds.[11,12,16,19,22,23] Here, we exploit this behavior to develop an unbiased, high-throughput screen (HTS) using GPMVs to identify new chemical modulators of rafts
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