QuiresThe Mitosome: Cardiolipin-Caspase-8-BidThe Mitosome: Cardiolipin-Caspase-8-BidFigure 4. Confocal microscopy study of the binding of Bid and caspase-8 to giant unilamellar vesicles containing cardiolipin. Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100:0) vesicles are presented in panels a to c and DOPC/CL (90:10) vesicles in panels d to f. Top: in a and d, protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05 of the hydrophobic dye DiO, as shown in (b, c) and in red, as shown in (e, f); bottom: overlay of green and red images (c, f). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and BidAlexa488, resulting in a non-fluorescent tBid. doi:10.1371/journal.pone.0055250.gcaspase-8 activation, or BAR [54] and FLASH, which mediate caspase-8 translocation to mitochondria [62,63,64], play an auxiliary role in the functional relationship between caspase-8 and CL. Possibly, MTCH2/MIMP [65] and its role in tBid recruitment may act in purchase 61177-45-5 synergy with CL-induced mitosome formation to 16985061 facilitate MOMP. The work we report here expands our knowledge of Bid-induced pro-apoptotic signalling and provides a description of the role of CL in capsase-8 recruitment and activation at the surface of the mitochondrial outer membrane. We 1527786 are however far from grasping all the intricateand complex molecular alterations and interactions that lead to the activation of Bid, mitochondrial membrane permeabilisation and apoptosis via the mitochondrial pathway following stimulation of the death receptors. The results that we present demonstrate and describe essential roles played by lipids in biological processes. In particular, they provide new insights into how mitochondrial specific lipids like CL can have active functions that go far beyond simply 256373-96-3 custom synthesis constituting a matrix for protein activities. Indeed, functional lipids appear to contribute not only to modulating the interactions between Bcl-Figure 5. Flow cytometric analysis of the interaction between CL-GUVs and caspase-8-Bid. (a ) Short-term effects (20 time points at 20 s intervals, total 6.66 min) of successive additions of procaspase-8 or Bid to GUVs-CL. (a ) Each product (caspase-8 or Bid) was added progressively, at 1.66-minute intervals, as shown in the recording, and the mean fluorescence of the vesicles was then measured. (a) Caspase-8 (Casp8) was added before Bid whereas, in (b), caspase-8 was added after three successive additions of Bid (10 nM, 40 nM and 60 nM). Even shortly after additions, the enzymatic system was functional, provided that caspase-8 bound to the giant unilamelar liposomes (GUVs). (c) the upper histogram, in black, corresponds to (a), and the lower histogram, in red, corresponds to (b); the occurrence of vesicles with a higher side scatter (SSC), due to procaspase8/Bid cleavage activity, was recorded and is plotted as a percentage ( ) of the total vesicle population. (d) The intensity of Bid-Alexa647 fluorescence associated with GUVs is shown as a function of procaspase-8 addition and time, for GUVs with (closed circles) or without (open circles) CL. doi:10.1371/journal.pone.0055250.gThe Mitosome: Cardiolipin-Caspase-8-BidFigure 6. Localised p.QuiresThe Mitosome: Cardiolipin-Caspase-8-BidThe Mitosome: Cardiolipin-Caspase-8-BidFigure 4. Confocal microscopy study of the binding of Bid and caspase-8 to giant unilamellar vesicles containing cardiolipin. Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100:0) vesicles are presented in panels a to c and DOPC/CL (90:10) vesicles in panels d to f. Top: in a and d, protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05 of the hydrophobic dye DiO, as shown in (b, c) and in red, as shown in (e, f); bottom: overlay of green and red images (c, f). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and BidAlexa488, resulting in a non-fluorescent tBid. doi:10.1371/journal.pone.0055250.gcaspase-8 activation, or BAR [54] and FLASH, which mediate caspase-8 translocation to mitochondria [62,63,64], play an auxiliary role in the functional relationship between caspase-8 and CL. Possibly, MTCH2/MIMP [65] and its role in tBid recruitment may act in synergy with CL-induced mitosome formation to 16985061 facilitate MOMP. The work we report here expands our knowledge of Bid-induced pro-apoptotic signalling and provides a description of the role of CL in capsase-8 recruitment and activation at the surface of the mitochondrial outer membrane. We 1527786 are however far from grasping all the intricateand complex molecular alterations and interactions that lead to the activation of Bid, mitochondrial membrane permeabilisation and apoptosis via the mitochondrial pathway following stimulation of the death receptors. The results that we present demonstrate and describe essential roles played by lipids in biological processes. In particular, they provide new insights into how mitochondrial specific lipids like CL can have active functions that go far beyond simply constituting a matrix for protein activities. Indeed, functional lipids appear to contribute not only to modulating the interactions between Bcl-Figure 5. Flow cytometric analysis of the interaction between CL-GUVs and caspase-8-Bid. (a ) Short-term effects (20 time points at 20 s intervals, total 6.66 min) of successive additions of procaspase-8 or Bid to GUVs-CL. (a ) Each product (caspase-8 or Bid) was added progressively, at 1.66-minute intervals, as shown in the recording, and the mean fluorescence of the vesicles was then measured. (a) Caspase-8 (Casp8) was added before Bid whereas, in (b), caspase-8 was added after three successive additions of Bid (10 nM, 40 nM and 60 nM). Even shortly after additions, the enzymatic system was functional, provided that caspase-8 bound to the giant unilamelar liposomes (GUVs). (c) the upper histogram, in black, corresponds to (a), and the lower histogram, in red, corresponds to (b); the occurrence of vesicles with a higher side scatter (SSC), due to procaspase8/Bid cleavage activity, was recorded and is plotted as a percentage ( ) of the total vesicle population. (d) The intensity of Bid-Alexa647 fluorescence associated with GUVs is shown as a function of procaspase-8 addition and time, for GUVs with (closed circles) or without (open circles) CL. doi:10.1371/journal.pone.0055250.gThe Mitosome: Cardiolipin-Caspase-8-BidFigure 6. Localised p.
