title: Probing lipid rafts with proximity imaging: actions of proatherogenic stimuli.
abstract: Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to cluster in microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years, several attempts have been made to use fluorescent probes to nondisruptively label these domains in living cells. Here, we have transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show colocalization of this fluoroprobe with another marker of lipid rafts, urokinase-type plasminogen activator receptor-1. ttGFP was used to quantify the cell surface area occupied by lipid rafts and to examine the effect of various proatherogenic signals on lipid rafts. Exposure of endothelial cells to asymmetric dimethylarginine and oxidized LDL (oxLDL), as well as oxidant stress, reduced the cell surface area occupied by lipid rafts. Next, the property of ttGFP to undergo a shift in absorbance depending on the clustering of these molecules was utilized to perform proximity imaging (PRIM). PRIM showed that nitric oxide (NO) increased the distance between GPI-anchored ttGFP molecules clustered in lipid-rich microdomains. This "unclustering" of GPI-anchored ttGFP was not reproduced by prooxidant signals and was due to reduction in membrane-cytoskeletal constraints on the lipid rafts. These findings suggested that two fundamentally different mechanisms modulate lipid rafts: 1) substance regulation of lipid rafts involving modification of cholesterol and sphingolipids and 2) structural regulation of lipid rafts through disruption of membrane-cytoskeletal interactions, switching off the spatial confinement of lipid rafts.
authors.affiliation: Department of Medicine, New York Medical Center, Valhalla, NY 10595, USA.
authors.forename: Susann
authors.initials: S
authors.lastname: Patschan
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Sergey
authors.initials: S
authors.lastname: Brodsky
authors.forename: David
authors.initials: D
authors.lastname: Sullivan
authors.forename: Dino A
authors.initials: DA
authors.lastname: De Angelis
authors.forename: Daniel
authors.initials: D
authors.lastname: Patschan
authors.forename: Michael S
authors.initials: MS
authors.lastname: Goligorsky
mesh_terms: D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D050197:Atherosclerosis / Q000503:physiopathology*; D002460:Cell Line; D008078:Cholesterol, LDL / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D042783:Endothelial Cells / Q000502:physiology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D021962:Membrane Microdomains / Q000502:physiology* / Q000648:ultrastructure*; D051379:Mice; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000096:biosynthesis; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
chemical_list: D000924:Anticholesteremic Agents; D008078:Cholesterol, LDL; D017261:Glycosylphosphatidylinositols; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D013481:Superoxides; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III