Y of the DOPC test membrane. On the contrary, the addition

Y of the DOPC test membrane. On the contrary, the addition of CL to a DOPC host membrane had a clear impact: it decreased the area expansion modulus and strongly decreased the overall sustainability of the membrane when subjected to mechanical stress, as demonstrated by the low value of the rupture tension tr. In the presence of tBid, the membrane stability, assessed as the expansion modulus Ks, returned to its initial value (Fig. 3b), but there was a further decrease in the rupture tension tr (Fig. 3c), to about 30 of the value initially obtained for the pure DOPC reference membrane. The simultaneous presence of KN-93 (phosphate) web caspase-8 and Bid resulted in similar values, whereas the addition of caspase-8 alone gave intermediate values.Confocal Microscopy Investigations of the Various Proteins Binding to GUVsConfocal microscopy provided evidence of an interaction between proteins and the test membranes. A multicolour approach was used: the membrane was labelled with DiD, shown in red in Fig. 4, with Bid shown in green. The images show the results of staining with the two dyes individually and the simultaneously obtained overlay image. BidGreen did not bind to get JNJ-7706621 vesicles containing phosphatidylcholine alone (Fig. 4a) or to CL-containing vesicles (not shown). These results are consistent with previous reports that Bid does not bind to either DOPC or DOPC/CL GUVs [33]. BidGreen did not bind to DOPC vesicles after the addition of caspase-8 (Fig. 4a), unless CL was also present (Fig. 4d). The binding of BidGreen to vesicles thus appeared to require the presence of both CL within the membrane and caspase-8 binding to it (Fig. 4d). The short-term effects of caspase8/BidGreen on CL-GUVs included not only complex binding (Fig. 4d), but also vesicle reorganisation and collapse (Fig. 4d ). The vesicles also displayed a significant decrease in green fluorescence (Fig. 4d) within a few minutes of addition of BidGreen. This decrease in fluorescence resulted from cleavage of the tagged BH1 H2 domain, the fluorophore remaining in the soluble p7 part of the protein after cleavage by capase-8 (as illustrated in Table 1). These observations provide evidence of a reaction platform, consisting of CL/caspase-8 and Bid, presenting an enzymatic activity. The CL-containing GUVs had a low rupture tension (Fig. 3c); they frequently broke and resealed, forming either smaller vesicles or, more often, irregularly shaped aggregates, due to the defective reorganisation of membrane material.Figure 2. Analysis of the effects of caspase-8, Bid and tBid on the Laurdan fluorescence of CL+ and CL2 liposomes. Generalised polarisation (GP, arbitrary units, a.u.) determined from Laurdan fluorescence measurements. GP values are reported for the various preparations, as described in the materials and methods. doi:10.1371/journal.pone.0055250.gmodified the GP considerably and the combination of Bid and 24786787 caspase-8 gave values similar to those obtained with tBid and caspase-8.Tension/rupture of Cardiolipin-containing Giant Unilamellar Vesicles (GUVs) in the Presence or Absence of Caspase-8 and Bid ProteinsThe effects of caspase-8, caspase-8+ Bid and/or tBid on membrane fluidity in Laurdan experiments (Fig. 2) suggest that the interaction of these proteins with a target membrane affects the elastic properties of the membrane. The elasticity theory for membranes is based on the theory of thin liquid films [42]. Two basic deformations can be identified: bending perpendicular to the bilayer surface, d.Y of the DOPC test membrane. On the contrary, the addition of CL to a DOPC host membrane had a clear impact: it decreased the area expansion modulus and strongly decreased the overall sustainability of the membrane when subjected to mechanical stress, as demonstrated by the low value of the rupture tension tr. In the presence of tBid, the membrane stability, assessed as the expansion modulus Ks, returned to its initial value (Fig. 3b), but there was a further decrease in the rupture tension tr (Fig. 3c), to about 30 of the value initially obtained for the pure DOPC reference membrane. The simultaneous presence of caspase-8 and Bid resulted in similar values, whereas the addition of caspase-8 alone gave intermediate values.Confocal Microscopy Investigations of the Various Proteins Binding to GUVsConfocal microscopy provided evidence of an interaction between proteins and the test membranes. A multicolour approach was used: the membrane was labelled with DiD, shown in red in Fig. 4, with Bid shown in green. The images show the results of staining with the two dyes individually and the simultaneously obtained overlay image. BidGreen did not bind to vesicles containing phosphatidylcholine alone (Fig. 4a) or to CL-containing vesicles (not shown). These results are consistent with previous reports that Bid does not bind to either DOPC or DOPC/CL GUVs [33]. BidGreen did not bind to DOPC vesicles after the addition of caspase-8 (Fig. 4a), unless CL was also present (Fig. 4d). The binding of BidGreen to vesicles thus appeared to require the presence of both CL within the membrane and caspase-8 binding to it (Fig. 4d). The short-term effects of caspase8/BidGreen on CL-GUVs included not only complex binding (Fig. 4d), but also vesicle reorganisation and collapse (Fig. 4d ). The vesicles also displayed a significant decrease in green fluorescence (Fig. 4d) within a few minutes of addition of BidGreen. This decrease in fluorescence resulted from cleavage of the tagged BH1 H2 domain, the fluorophore remaining in the soluble p7 part of the protein after cleavage by capase-8 (as illustrated in Table 1). These observations provide evidence of a reaction platform, consisting of CL/caspase-8 and Bid, presenting an enzymatic activity. The CL-containing GUVs had a low rupture tension (Fig. 3c); they frequently broke and resealed, forming either smaller vesicles or, more often, irregularly shaped aggregates, due to the defective reorganisation of membrane material.Figure 2. Analysis of the effects of caspase-8, Bid and tBid on the Laurdan fluorescence of CL+ and CL2 liposomes. Generalised polarisation (GP, arbitrary units, a.u.) determined from Laurdan fluorescence measurements. GP values are reported for the various preparations, as described in the materials and methods. doi:10.1371/journal.pone.0055250.gmodified the GP considerably and the combination of Bid and 24786787 caspase-8 gave values similar to those obtained with tBid and caspase-8.Tension/rupture of Cardiolipin-containing Giant Unilamellar Vesicles (GUVs) in the Presence or Absence of Caspase-8 and Bid ProteinsThe effects of caspase-8, caspase-8+ Bid and/or tBid on membrane fluidity in Laurdan experiments (Fig. 2) suggest that the interaction of these proteins with a target membrane affects the elastic properties of the membrane. The elasticity theory for membranes is based on the theory of thin liquid films [42]. Two basic deformations can be identified: bending perpendicular to the bilayer surface, d.