Key Points Granzymes diffuse through perforin pores on the target cell plasma membrane.

Melanoma cells break down lysophosphatidic acid from the environment, creating a chemotactic gradient that the tumor cells then follow; this provides an explanation for the rapid metastasis of melanoma.

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.

Granzymes (Grz) are a family of serine proteases found in the granules of cytotoxic lymphocytes and are emerging as an important group of proteins involved in immune function and surveillance. Grz have both cytotoxic and more recently reported non-cytotoxic roles, however these functions are still subject to thorough investigation. The significance of the cytotoxic and importantly the non-cytotoxic roles of Grz will be discussed in this review, detailing accepted and controversial functions.

Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.