Isolation, characterisation and reconstitution of cell death signalling complexes.

Apoptosis and necroptosis are dependent on the formation/activation of distinct multi-protein complexes; these include the Death-Inducing Signalling Complex (DISC), apoptosome, piddosome, necrosome and ripoptosome. Despite intense research, the mechanisms that regulate assembly/function of several of these cell death signalling platforms remain to be elucidated. It is now increasingly evident that the composition and stoichiometry of components within these key signalling platforms not only determines the final signalling outcome but also the mode of cell death. Characterising these complexes can therefore provide new insights into how cell death is regulated and also how these cell death signalling platforms could potentially be targeted in the context of disease. Large multi-protein complexes can initially be separated according to their size by gel filtration or sucrose density gradient centrifugation followed by subsequent affinity-purification or immunoprecipitation. The advantage of combining these techniques is that you can assess the assembly of individual components into a complex and then assess the size and stoichiometric composition of the native functional signalling complex within a particular cell type. This, alongside reconstitution of a complex from its individual core components can therefore provide new insight into the mechanisms that regulate assembly/function of key multi-protein signalling complexes. Here, we describe the successful application of a range of methodologies that can be used to characterise the assembly of large multi-protein complexes such as the apoptosome, DISC and ripoptosome. Together with their subsequent purification and/or reconstitution, these approaches can provide novel insights into how cell death signalling platforms are regulated in both normal cell physiology and disease.

Immunomagnetic isolation of tumor necrosis factor receptosomes.

Internalized tumor necrosis factor (TNF) receptor-1 (TNF-R1) recruits the adaptor proteins TRADD and FADD, as well as caspase-8, to establish the "death-inducing signaling complex" (DISC). DISC formation and apoptosis depend strictly on TNF-R1 internalization, whereas recruitment of TRAF-2 and RIP-1 to signal for NF-kappaB activation occurs from TNF-R1 at the cell surface. Findings revealed that TNF-R1 establishes divergent TNF signaling pathways depending on compartmentalization of TNF-R1 to the plasma membrane or to plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC. These data were obtained by a novel technique for the isolation of morphologically intact endocytic vesicles containing magnetically labeled TNF-R1 complexes (termed TNF receptosomes) using a custom-made high gradient magnetic chamber. This chapter describes the protocol of immunomagnetic labeling using biologically active biotin TNF as a ligand coupled to magnetic streptavidin nanobeads, followed by a gentle mechanical homogenization procedure to preserve the morphological structure of membrane vesicles containing activated TNF-R1 complexes. Isolation of the magnetized receptosomes in a high magnetic gradient is described, and the kinetics of TNF-R1 internalization and endosomal trafficking/maturation of the receptosomes is characterized. Using a biotinylated anti-CD95 antibody as ligand and streptavidin-coated magnetic nanobeads for separation in the high gradient magnetic chamber, the immunomagnetic separation approach was additionally applied to characterize the internalization and maturation of CD95 receptosomes.