Fas signaling induces raft coalescence that is blocked by cholesterol depletion in human RPE cells undergoing apoptosis

Investigative Ophthalmology & Visual Science
James E LincolnLawrence S Morse

Abstract

To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)-labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-microm-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raft-associated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-microm vesicles with both GM1 ...Continue Reading

Citations

Apr 25, 2007·Apoptosis : an International Journal on Programmed Cell Death·Walter MalorniMaurizio Sorice
May 18, 2010·Journal of Lipid Research·Daniel A BricarelloAtul N Parikh
Jan 1, 2010·The Journal of Investigative Dermatology·Florian SpörlAnnika Schrader
May 1, 2012·Integrative Biology : Quantitative Biosciences From Nano to Macro·Huawen WuYin Yeh
Oct 13, 2007·The Journal of Biological Chemistry·Ehssan Sharif-AskariRafick-Pierre Sékaly
May 25, 2010·Archives of Biochemistry and Biophysics·Witold K SubczynskiJustyna Widomska

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis