Late Endosomal Degradation of Low-Density Lipoprotein Probed with Multi-Color Single Particle Tracking Fluorescence Microscopy

Abstract

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular membrane biosynthesis. Using multi-color single particle tracking fluorescence microscopy, we have probed the intracellular degradation of low-density lipoprotein in living cells. The unique aspect of our experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles are labeled with multiple fluorophores resulting in a quenched fluorescent signal. Control experiments demonstrate that enzymatic degradation of the LDL particle results in an increase in fluorescence. The ability to directly observe LDL degradation allows us to determine which vesicle is responsible for degradation and quantify the vesicle dynamics involved in LDL degradation. Visualization of early endosomes, late endosomes and lysosomes is accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle are measured simultaneously. The measured colocalization durations are then correlated with changes in fluorescence intensity due to LDL degradation. We observe that degradation of LDL occurs in the late endosome. While there are a broad distribution of colocalization durations of LDL with Rab7, a late endosomal protein, only relatively long (>420 s) colocalization leads to the degradation of LDL. These studies, which are the first to directly observe the degradation of LDL within a cell, support a model in which late endosomes are the site of degradation with lysosomes serving as enzyme storage vesicles.