title: Detection of apoptosis through the lipid order of the outer plasma membrane leaflet.
abstract: Cell plasma membranes of living cells maintain their asymmetry, so that the outer leaflet presents a large quantity of sphingomyelin, which is critical for formation of ordered lipid domains. Here, a recently developed probe based on Nile Red (NR12S) was applied to monitor changes in the lipid order specifically at the outer leaflet of cell membranes. Important key features of NR12S are its ratiometric response exclusively to lipid order (liquid ordered vs. liquid disordered phase) and not to surface charge, the possibility of using it at very low concentrations (10-20nM) and the very simple staining protocol. Cholesterol extraction, oxidation and sphingomyelin hydrolysis were found to red shift the emission spectrum of NR12S, indicating a decrease in the lipid order at the outer plasma membrane leaflet. Remarkably, apoptosis induced by three different agents (actinomycin D, camptothecin, staurosporine) produced very similar spectroscopic effects, suggesting that apoptosis also significantly decreases the lipid order at this leaflet. The applicability of NR12S to detect apoptosis was further validated by fluorescence microscopy and flow cytometry, using the ratio between the blue and red parts of its emission band. Thus, for the first time, an environment-sensitive probe, sensitive to lipid order, is shown to detect apoptosis, suggesting a new concept in apoptosis sensing.
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France.
authors: Zeinab
authors: Z
authors: Darwich
authors: Andrey S
authors: AS
authors: Klymchenko
authors: Oleksandr A
authors: OA
authors: Kucherak
authors: Ludovic
authors: L
authors: Richert
authors: Yves
authors: Y
authors: Mély
mesh_terms: D017209:Apoptosis; D002166:Camptothecin / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010078:Oxazines; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000737:chemistry*; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008055:Lipids; D010078:Oxazines; D013109:Sphingomyelins; D003609:Dactinomycin; D002784:Cholesterol; D019311:Staurosporine; C044808:nile red; D002166:Camptothecin