title: Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi.
abstract: Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.
authors.affiliation: Departments of Medicine and Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Peyrot
authors.affiliation: Biochemistry, Stanford University School of Medicine, Stanford, California 94305, and.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Nachtergaele
authors.affiliation: Biochemistry, Stanford University School of Medicine, Stanford, California 94305, and.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Luchetti
authors.affiliation: Departments of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Laurel K
authors.initials: LK
authors.lastname: Mydock-McGrane
authors.affiliation: The Diabetic Cardiovascular Research Center, and Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Hideji
authors.initials: H
authors.lastname: Fujiwara
authors.affiliation: The Diabetic Cardiovascular Research Center, and Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: David
authors.initials: D
authors.lastname: Scherrer
authors.affiliation: Departments of Cell Biology and Physiology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Andrew
authors.initials: A
authors.lastname: Jallouk
authors.affiliation: Departments of Cell Biology and Physiology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Schlesinger
authors.affiliation: The Diabetic Cardiovascular Research Center, and Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Ory
authors.affiliation: Departments of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110; Departments ofAnesthesiology, and Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110; Departments of Psychiatry, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110; The Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110.
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.affiliation: Departments of Medicine and Stanford University School of Medicine, Stanford, California 94305; Biochemistry, Stanford University School of Medicine, Stanford, California 94305, and. Electronic address: rrohatgi@stanford.edu.
authors.forename: Rajat
authors.initials: R
authors.lastname: Rohatgi
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D016466:CHO Cells; D057930:Click Chemistry; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells
chemical_list: D005456:Fluorescent Dyes; D006888:Hydroxycholesterols
keywords: Imaging; Lipids; Lysosomes; Oxysterol; Signaling; Trafficking