title: Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking.
abstract: The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells. The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.
authors.affiliation: Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. kruthh@nhlbi.nih.gov
authors.forename: H S
authors.initials: HS
authors.lastname: Kruth
authors.forename: I
authors.initials: I
authors.lastname: Ifrim
authors.forename: J
authors.initials: J
authors.lastname: Chang
authors.forename: L
authors.initials: L
authors.lastname: Addadi
authors.forename: D
authors.initials: D
authors.lastname: Perl-Treves
authors.forename: W Y
authors.initials: WY
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004951:Esterification; D005312:Fetal Blood; D005347:Fibroblasts / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000648:ultrastructure; D008563:Membrane Lipids / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors
chemical_list: D000911:Antibodies, Monoclonal; D002788:Cholesterol Esters; D004791:Enzyme Inhibitors; D008077:Lipoproteins, LDL; D008563:Membrane Lipids; C032629:acetyl-LDL; D002784:Cholesterol; D002785:Sterol O-Acyltransferase