Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

SummaryCytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC . Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing . In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules . Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTUTC interaction . On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded . The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-independent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.

CD95 is the quintessential death receptor and, when it is bound by ligand, cells undergo apoptosis. Recent evidence suggests, however, that CD95 mediates not only apoptosis but also diverse nonapoptotic functions depending on the tissue and the conditions.

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Upon DNA damage, a complex called the PIDDosome is formed and either signals NF-jB activation and thus cell survival or alternatively triggers caspase-2 activation and apoptosis. PIDD (p53-induced protein with a death domain) is constitutively processed giving rise to a 48-kDa N-terminal fragment containing the leucine-rich repeats (LRRs, PIDD-N) and a 51-kDa C-terminal fragment containing the death domain (DD, PIDD-C). The latter undergoes further cleavage resulting in a 37-kDa fragment (PIDD-CC). Here we show that processing occurs at S446 (generating PIDD-C) and S588 (generating PIDD-CC) by an auto-processing mechanism similar to that found in the nuclear pore protein Nup98/96 and inteins. Auto-cleavage of PIDD determines the outcome of the downstream signaling events. Whereas initially formed PIDD-C mediates the activation of NF-jB via the recruitment of RIP1 and NEMO, subsequent formation of PIDD-CC causes caspase-2 activation and thus cell death. A non-cleavable PIDD mutant is unable to translocate from the cytoplasm to the nucleus and loses both activities. In this way, autoproteolysis of PIDD might participate in the orchestration of the DNA damage-induced life and death signaling pathways.