Live-cell imaging and lipidomics of low density lipoprotein containing intrinsically fluorescent cholesteryl esters

Low density lipoprotein (LDL) delivers cholesterol to cells in the body in the form of cholesteryl esters (CEs), and dysfunction of this pathway is associated with various diseases. Due to the lack of suitable tools, our understanding of the intracellular transport and hydrolysis of CEs is limited. We present a novel approach for studying LDL-derived CEs in cells, using fatty acyl chain conjugates of the intrinsically fluorescent cholestatrienol (CTL). We demonstrate that CTL esters reconstituted into LDL particles are hydrolyzed in late endosomes and lysosomes (LE/LYSs) by acid lipase, while an LDL-derived CTL ether analog cannot leave LE/LYSs. Using live-cell imaging, lipidomics, and multimodal Bayesian modeling, we discover a sequential biphasic transport of LDL-derived CTL to LE/LYSs matching the kinetics of the lysosomal hydrolysis of LDL-associated CTL-ester with a half-time of 3.0 hours. Hydrolyzed CTL derived from LDL-associated CTL esters is rapidly re-esterified with a similar half-time and stored in lipid droplets, demonstrating efficient sterol transport to the endoplasmic reticulum (ER). The latter is supported by the detection of a faint CTL staining in the ER and by extensive contact formation between endo-lysosomes containing LDL and ER tubules. Using lipidomics and Bayesian kinetic modeling, we also track LDL-derived CEs and triacylglycerols in cells and determine the uptake kinetics for each lipid species individually. Our novel approach allows for precise measurement of post-endocytic trafficking and metabolism of LDL-derived cholesterol and other lipids in living cells.