title: Intracellular degradation of low-density lipoprotein probed with two-color fluorescence microscopy.
abstract: The intracellular vesicle-mediated degradation of extracellular cargo is an essential cellular function. Using two-color single particle tracking fluorescence microscopy, we have probed the intracellular degradation of low-density lipoprotein (LDL) in living cells. To detect degradation, individual LDL particles were heavily labeled with multiple fluorophores resulting in a quenched fluorescent signal. The degradation of the LDL particle then resulted in an increase in fluorescence. Endocytic vesicles were fluorescently labeled with variants of GFP. We imaged the transient colocalization of LDL with endocytic vesicles while simultaneously measuring the intensity of the LDL particle as an indicator of degradation. These studies demonstrate that late endosomes are active sites of degradation for LDL. Measurement of the time from colocalization with lysosome-associated membrane protein 1 (LAMP1) vesicles to degradation suggests that LAMP1-vesicles initiate the degradative event. Observing degradation as it occurs in living cells makes it possible to describe the complete endocytic pathway of LDL from internalization to degradation. More generally, this research provides a model for the intracellular degradation of extracellular cargo and a method for its study in living cells.
authors.affiliation: Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, 30332, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Humphries
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Fay
authors.forename: Christine K
authors.initials: CK
authors.lastname: Payne
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D002401:Cathepsin B / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D021381:Protein Transport / Q000187:drug effects; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000730:Androstadienes; D002232:Carbocyanines; D008077:Lipoproteins, LDL; D008164:Luminescent Proteins; D052118:Lysosomal-Associated Membrane Protein 1; D000090802:rab7 GTP-Binding Proteins; C024286:3,3'-dioctadecylindocarbocyanine; D014357:Trypsin; D002401:Cathepsin B; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000077191:Wortmannin