Giant unilamellar vesicles (GUVs) as a new tool for analysis of caspase-8/Bid-FL complex binding to cardiolipin and its functional activity

O Jalmar,L Berland,F Gonzalvez,P X Petit,A J García-Sáez

doi:10.1038/cddis.2010.81

O Jalmar, L Berland + Show 3 more

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https://doi.org/10.1038/cddis.2010.81

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Cardiolipin (CL) has recently been shown to be both an anchor and an essential activating platform for caspase-8 on mitochondria. These platforms may be at the mitochondrial contact sites in which truncated Bid (tBid) has been demonstrated to be located. A possible role for CL is to anchor caspase-8 at contact sites (between inner and outer membranes), facilitating its self-activation, Bid-full length (FL) cleavage, tBid generation (and Bax/Bak activation and oligomerization), mitochondrial destabilization and apoptosis. We have developed an in vitro system that mimics the mitochondrial membrane contact site platform. This system involves reconstituting caspase-8, Bid-FL and CL complexes in giant unilamellar vesicles (GUVs). We first validated the system by flow cytometry analysis of light-scattering properties and nonyl acridine orange staining of their CL content. Then, we used flow cytometry analysis to detect the binding of active caspase-8 to CL and the subsequent truncation of bound Bid-FL. The tBid generated interacts with CL and induces GUV breakage and partial re-vesiculation at a smaller size. Our findings suggest an active role for mitochondrial membrane lipids, particularly CL, in binding active caspase-8 and providing a docking site for Bid-FL. This phenomenon was previously only poorly documented and substantially underestimated.

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In type I cells, Fas-induced activation of caspase-8 leads to activation of effector caspases and subsequently cell death
We recently reported that Fas receptor activation induced the translocation of active caspase-8 to the mitochondrial surface, a step that was dependent on the presence of mature CL in the outer membrane (OMM).[21]
We investigated the binding and effects of truncated Bid (tBid) on giant unilamellar vesicles (GUVs) containing or not containing CL. tBid did not bind to vesicles containing only phosphatidylcholine (Figure 2a, b, e and f), but showed specific binding to GUVs containing CL ((Figure 2c, d, g and h)

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In type I cells, Fas-induced activation of caspase-8 leads to activation of effector caspases and subsequently cell death. This is consistent with previous findings concerning CL-mediated selectivity of tBid interactions with lipid membranes.[16,22,25,26,27,28] The weak non-specific binding did not modify the light scatter or the fluorescence of PC-only-GUVs (Figure 2b and f).

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