FRET‐based Assay Measures the Organization of LDL‐LDL‐R Complexes During LDL Trafficking

Abstract

Low Density Lipoprotein (LDL) is the primary mode of cholesterol transport in the bloodstream. Blood LDL enters liver cells via LDL-receptor (LDL-R)-mediated endocytosis and is delivered to late endosomes/lysosomes, where low pH induces the release of LDL. Upon LDL degradation, cholesterol is released for cellular uptake. Here we study the sub-cellular organization of LDL during LDL-R-mediated trafficking. We have developed a novel Fluorescence Resonance Energy Transfer (FRET)-based assay that investigates the organization of fluorescently labeled LDL during internalization, release and breakdown. MDCK epithelial cells are loaded with LDL conjugated to donor or acceptor fluorophores at 37oC and confocal microscopy is used to collect multi-channel FRET images. Custom software was developed to automatically process FRET images and reduce image reading bias. The automated ROI selection developed in this project is capable of accurately identifying fluorescent ROIs. Results show correlation between the energy transfer (E%) and acceptor levels; a direct relationship detected 30 minutes post internalization indicates a LDL random organization in late endosomes. Agents that slow LDL release generate a clustered LDL organization. As an in vivo assay for LDL release from LDL-R, the package developed holds potential in screening agents that modify LDL and cholesterol trafficking.