NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing.

Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells. Apoptosis is coordinated by the activity of initiator and effector caspases.1-4 While effector caspases are dimeric zymogens that become activated by cleavage, initiator caspases are normally expressed as monomeric zymogens and their activity is initiated by dimerization in a multimeric complex. 5,6 The initiator caspase procaspase-8 dimerizes in the death-inducing signaling complex (DISC) formed around activated death receptors. 7 In the context of CD95, the DISC contains clustered receptors bound to the adaptor protein FADD. FADD can recruit several proteins, 8,9 including procaspase-8 and -10 through their prodomain. In type I cells, active caspase-8/10 directly cleaves and activates effector caspase-3/-7, thus inducing apoptosis. In type II cells, this activation is blocked by XIAP, but cleavage of BID by active caspase-8/10 induces mitochondrial outer membrane permeabilization (MOMP), followed by initiator caspase-9 activation and release of XIAP-inhibitor SMAC, leading to massive caspase-3/7 activity. 10,11 Biochemical and structural studies showed that dimerization but also cleavage in the catalytic subunit of caspase-8 is required for efficient cleavage of caspase-3 and BID and for apoptosis.12-14 Supporting this, the non-cleavable D387A caspase-8 mutant compromises apoptosis in mice. 15Although caspase-8 dimerization generates some activity, cleavage in the catalytic units likely stabilizes the active form and increases activity. 13,16 Caspase-8 is also cleaved between the prodomain and the catalytic unit of the enzyme, at D210 and D216, 17,18 and subsequently released from the DISC. Active caspase-8 can be detected on the plasma membrane, before release, with fluorescent inhibitors. 19 In contrast, by designing a procaspase-8 artificially dimerized on the plasma membrane, Martin et al.18 suggested that the cytosolic release is necessary to cleave caspase-3 and BID. Other studies proposed that fully processed ...

The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.