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.