Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level

Radošević, Katarina; Grooth, B.G. de; Greve, Jan

doi:10.1002/cyto.990200403

Cited by 10 publications

(9 citation statements)

References 31 publications

(20 reference statements)

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“…8d). For K562 cells, an average pH i of 6.9 -7.2 at pH e values of 6.8 -7.6 has been reported (25). Our data show that if extracellular pH declined below 6.8, the pH i of K562 and Daudi cells could not further be regulated and decreased to more acidic values.…”

Section: Figcontrasting

confidence: 54%

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Acidic pH Inhibits Non-MHC-Restricted Killer Cell Functions

Fischer

Müller

Fischer

et al. 2000

Clinical Immunology

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Section: Figcontrasting

confidence: 54%

“…To determine the influence of pH e on pH i during a 4-h incubation period reflecting the conditions of the cytotoxic assays, we measured the pH i of target and effector cells separately using the fluorescent pH indicator BCECF (23)(24)(25).…”

Section: Measurement Of Ph I By Flow Cytometrymentioning

confidence: 99%

Acidic pH Inhibits Non-MHC-Restricted Killer Cell Functions

Fischer

Müller

Fischer

et al. 2000

Clinical Immunology

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“…4 Subsequent passage to the cytosol (presumably through ring-like pores) is then thought to enable the granzymes to elicit cell death. 12 Here, using a modified quantitative flow-based approach to measure Ca 2 þ flux at the single cell level, 13 we show that non-necrotic target cells that became apoptotic in the presence of GzmB show rapid (o15 sec) albeit minimal (70-120 nM) Ca 2 þ influx (Figures 1a and b; Figures 3b and c). Importantly, 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (BAPTA-AM), an intracellular Ca 2 þ chelator, did not modify susceptibility to GzmB-induced apoptosis.…”

Section: Resultsmentioning

confidence: 74%

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

et al. 2014

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Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets. Cell Death and Differentiation ( The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation. 1 Ring-shaped transmembrane PFN pores hereafter called 'cylindrical pores', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis. [2][3][4] In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal, 5 that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding. 6 This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids. [6][7][8] In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and t...

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“…The subset of interest, that is, target cells prepared for granzyme delivery, resides among the apparently normal population. As the concentration of PFN increases, the percentage of necrotic cells rises until all the cells die presumably due to the formation of enough pores to cause a massive Ca influx [29]. To our knowledge, markers that identify the PFN-induced membrane changes that might be associated with granzyme delivery have not been described.…”

Section: Resultsmentioning

confidence: 99%

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

et al. 2011

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The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

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Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level

Cited by 10 publications

References 31 publications

Acidic pH Inhibits Non-MHC-Restricted Killer Cell Functions

Acidic pH Inhibits Non-MHC-Restricted Killer Cell Functions

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

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