Probing the Intracellular Reaction Dynamics of Low Density Lipoprotein Using Single Particle Tracking Fluorescence Microscopy

Abstract

Interactions between substrate-containing late endosomes and enzyme-containing lysosomes mediate intracellular reactions that lead to the degradation of the substrate. To monitor the intracellular degradation of low density lipoprotein (LDL) in live cells, individual LDL particles were labeled with approximately 200 lipophilic, fluorescent dye molecules. Due to the close proximity of individual fluorophores, the emission of photons by the fluorophore is quenched. Upon enzymatic degradation, the LDL particle exhibits a 3-fold fluorescence enhancement. The change in fluorescence intensity of the LDL particle is observed with fluorescence microscopy and analyzed using single particle tracking. Dequenching events are observed as a single-step increase in intensity. Wortmannin, a drug that inhibits late endosome fusion, is used to block the transport of LDL to the lysosome. Individual dequenching events are not observed in wortmannin-treated cells suggesting that degradation of the LDL particle requires late endosome-lysosome interactions. This LDL labeling scheme will be extended to two-color single particle tracking experiments to determine the fraction of late endosome-lysosome interactions that lead to enzymatic degradation of LDL particles.