Indicated on the graphs. The liposomes, suspended in light buffer (100 mM NaCl, 2 mM MgCl2 and 10 mM Tris-HCl, pH 7.1), were centrifuged for 1 h at 55,000 rpm in a Beckman SW 70.1 rotor at 10uC.Binding of Bid and P7C3 web caspase-8 to LiposomesPelleted liposomes were obtained as described above (the liposome mixture was shiny and free of aggregates); 50 ml of the precipitate was resuspended in 450 ml of light buffer and incubated with either caspase-8 (290 nM) or Bid (50 nM) for flow cytometry analysis.Immunoblot AnalysisThe liposome pellet was lysed in 1 sodium cholate, resuspended in a protein sample buffer containing DTT, and resolved by SDS-PAGE in a 4?2 polyacrylamide gel (NuPAGE). The proteins were transferred to a membrane and pThe Mitosome: Cardiolipin-Caspase-8-BidFigure 1. Binding of Bid and caspase-8 to CL-containing large unilamellar liposomes (LUVs). (a) Schematic diagram of caspase-8 autoprocessing during Fas-mediated apoptosis. Upon dimerisation, procaspase-8 (p55) is initially cleaved between its two active subunits, p18 and p10, to generate the p43/p10 heterodimer; p43 is then cleaved between the death effector domain (DED) and the p18 subunit, to produce the fully active p18/p10 form. (b) Western blot analysis of caspase-8 binding to the “contact site mimetic” liposomes or similar liposomes without CL, in which the CL was replaced with PE (22 ) (c) Caspase-8 binding, as detected by caspACE FITC-VAD-fmk binding to the active site, to liposomes of various compositions (monolipid liposomes made from PA, PC, PE, PI, PG or cholesterol, and mixed liposomes composed of DOPC+CL, DOPC+PE, DOPC+CL+PE at various molar ratios, contact site mimetic liposomes; for details see materials and methods). (d) Flow cytometric analysis of CL+ and DOPC-only liposomes in the presence or absence of BidAlexa488. The black spectrum correspond to control vesicles whereas the red spectrum correspond to the vesicles plus BidAlexa488. The blue spectrum results from an alkaline wash of the CL+ liposomes. The alkaline wash involved centrifugation of liposomes and resuspending them in 0.1 M Na2CO3, pH 11.5. The liposomes were then analysed directly by flow cytometry. Fm: fluorescence mean value, in arbitrary units (a.u.). doi:10.1371/journal.pone.0055250.gand p43 bands were detected with anti-Licochalcone-A biological activity caspase antibodies directed against the DED domain of caspase 8 (Becton-Dickinson).Preparation of Giant Unilamellar VesiclesThe GUVs consisted mostly of DOPC and CL, with CL content ranging from 0 to 20 (mol/mol), as indicated in the figure legends. All lipid mixtures were prepared in chloroform stock 1662274 solution, at a total concentration of 1 mg/ml, with the appropriatelipid DOPC/CL ratio. Vesicles were grown in sucrose solutions (300 mOsm). For confocal microscopy, GUVs were prepared by the electro-swelling method [36]. We spread 5 ml of lipid mixture (1 mg ml21 in chloroform) directly onto two Pt wire 1317923 electrodes kept 1 cm apart in a swelling chamber. The chamber was filled with swelling solution (300 mM sucrose) and the wires were connected to a power generator; a voltage of 2.3 V at 10 Hz was applied for 1 h at room temperature, for the field-supportedThe Mitosome: Cardiolipin-Caspase-8-Bidswelling of GUVs from the lipid films. The GUVs were then detached from the electrodes by increasing the frequency to 2 kHz for 30 min. Finally, they were carefully harvested with a syringe with a large-diameter needle. For flow cytometry, GUVs were prepared by the electroformatio.Indicated on the graphs. The liposomes, suspended in light buffer (100 mM NaCl, 2 mM MgCl2 and 10 mM Tris-HCl, pH 7.1), were centrifuged for 1 h at 55,000 rpm in a Beckman SW 70.1 rotor at 10uC.Binding of Bid and Caspase-8 to LiposomesPelleted liposomes were obtained as described above (the liposome mixture was shiny and free of aggregates); 50 ml of the precipitate was resuspended in 450 ml of light buffer and incubated with either caspase-8 (290 nM) or Bid (50 nM) for flow cytometry analysis.Immunoblot AnalysisThe liposome pellet was lysed in 1 sodium cholate, resuspended in a protein sample buffer containing DTT, and resolved by SDS-PAGE in a 4?2 polyacrylamide gel (NuPAGE). The proteins were transferred to a membrane and pThe Mitosome: Cardiolipin-Caspase-8-BidFigure 1. Binding of Bid and caspase-8 to CL-containing large unilamellar liposomes (LUVs). (a) Schematic diagram of caspase-8 autoprocessing during Fas-mediated apoptosis. Upon dimerisation, procaspase-8 (p55) is initially cleaved between its two active subunits, p18 and p10, to generate the p43/p10 heterodimer; p43 is then cleaved between the death effector domain (DED) and the p18 subunit, to produce the fully active p18/p10 form. (b) Western blot analysis of caspase-8 binding to the “contact site mimetic” liposomes or similar liposomes without CL, in which the CL was replaced with PE (22 ) (c) Caspase-8 binding, as detected by caspACE FITC-VAD-fmk binding to the active site, to liposomes of various compositions (monolipid liposomes made from PA, PC, PE, PI, PG or cholesterol, and mixed liposomes composed of DOPC+CL, DOPC+PE, DOPC+CL+PE at various molar ratios, contact site mimetic liposomes; for details see materials and methods). (d) Flow cytometric analysis of CL+ and DOPC-only liposomes in the presence or absence of BidAlexa488. The black spectrum correspond to control vesicles whereas the red spectrum correspond to the vesicles plus BidAlexa488. The blue spectrum results from an alkaline wash of the CL+ liposomes. The alkaline wash involved centrifugation of liposomes and resuspending them in 0.1 M Na2CO3, pH 11.5. The liposomes were then analysed directly by flow cytometry. Fm: fluorescence mean value, in arbitrary units (a.u.). doi:10.1371/journal.pone.0055250.gand p43 bands were detected with anti-caspase antibodies directed against the DED domain of caspase 8 (Becton-Dickinson).Preparation of Giant Unilamellar VesiclesThe GUVs consisted mostly of DOPC and CL, with CL content ranging from 0 to 20 (mol/mol), as indicated in the figure legends. All lipid mixtures were prepared in chloroform stock 1662274 solution, at a total concentration of 1 mg/ml, with the appropriatelipid DOPC/CL ratio. Vesicles were grown in sucrose solutions (300 mOsm). For confocal microscopy, GUVs were prepared by the electro-swelling method [36]. We spread 5 ml of lipid mixture (1 mg ml21 in chloroform) directly onto two Pt wire 1317923 electrodes kept 1 cm apart in a swelling chamber. The chamber was filled with swelling solution (300 mM sucrose) and the wires were connected to a power generator; a voltage of 2.3 V at 10 Hz was applied for 1 h at room temperature, for the field-supportedThe Mitosome: Cardiolipin-Caspase-8-Bidswelling of GUVs from the lipid films. The GUVs were then detached from the electrodes by increasing the frequency to 2 kHz for 30 min. Finally, they were carefully harvested with a syringe with a large-diameter needle. For flow cytometry, GUVs were prepared by the electroformatio.
