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This Article Full Text Full Text (PDF) All Versions of this Article: 290/6/H2210    most recent 01112.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Patschan, S. Articles by Goligorsky, M. S. Search for Related Content PubMed PubMed Citation Articles by Patschan, S. Articles by Goligorsky, M. S.

Probing lipid rafts with proximity imaging: actions of proatherogenic stimuli

¹Departments of Medicine and Pharmacology, New York Medical Center, Valhalla; ²Department of Medicine, State University of New York, Stony Brook; and ³Memorial Sloan-Kettering Cancer Center, New York, New York

Submitted 24 October 2005 ; accepted in final form 10 December 2005

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.

nitric oxide; endothelium; urokinase-type plasminogen activator receptor; oxidized low-density lipoprotein

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