Caspase 8 Activity in Membrane Blebs After Anti-Fas Ligation

Abstract

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry

“…As will be shown initially in this paper, this is also true of caspase activation as measured by flow cytometry. Nevertheless, we have observed microscopically what may be very early apoptotic cells following labeling for caspase activation; these cells show very low levels of nonuniform, punctate, and highly localized caspase activity (18). This is consistent with other studies that suggest nonuniform, organelle-specific caspase localization during the early stages of caspase cascade activation (19,20).…”

“…Previous confocal microscopy of PhiPhiLux-positive cells has demonstrated early apoptotic cells with nonuniform, punctate caspase activity apparently localized to the cytoplasm and likely compartmentalized in vesicles (18). Traditional peak or area analysis by flow cytometry was not sufficiently sensitive to detect this early caspase activation.…”

“…Nevertheless, previous analyses of caspase-active cells by confocal microscopy has suggested that intermediate stages of caspase activation may be observable (18). Previous confocal microscopy of PhiPhiLux-positive cells has demonstrated early apoptotic cells with nonuniform, punctate caspase activity apparently localized to the cytoplasm and likely compartmentalized in vesicles (18).…”

“…Zeiosis is a hallmark of apoptosis as defined by morphological analysis. Nevertheless, it has received little attention from many apoptotic studies due to the lack of a good quantitative method for detection (18).…”

Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

“…Once a substrate is recognized and cleaved by its cognate protease, the cleaved fragments are trapped on the side of the membrane where the protease activity resides [30,31]. (There is a low, but finite, diffusion rate of fragments across membranes.)…”