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

Jalmar, O.; García-Sáez, Ana J.; Berland, L.; Gonzalvez, F.; Petit, P. X.

doi:10.1038/cddis.2010.81

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

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Jalmar, O., García-Sáez, A. J., Berland, L., Gonzalvez, F., & Petit, P. X. (2010). Giant unilamellar vesicles (GUVs) as a new tool for analysis of caspase-8/Bid-FL complex binding to cardiolipin and its functional activity. Cell Death and Disease, 1: e103. doi:10.1038/cddis.2010.81.

Cite as: https://hdl.handle.net/21.11116/0000-000F-E2D2-4

Abstract

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.