The Targeting of Plasmalemmal Ceramide to Mitochondria during Apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1—a member of a family of Ca2+ and membrane-binding proteins—to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this “kiss-of-death” increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis

Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks

Microvesicle Shedding and Lysosomal Repair Fulfill Divergent Cellular Needs during the Repair of Streptolysin O-Induced Plasmalemmal Damage

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens

Ca²⁺-dependent repair of pneumolysin pores:A new paradigm for host cellular defense against bacterial pore-forming toxins

AbstractPneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood.We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance.The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects.Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections. This article is part of a Special Issue entitled: 13th European Symposium on Calcium

SLO-perforation inflicts mechanical damage and triggers lysosomal fusion.

<p>(<b>A</b>) HEK 293 cells do not adhere extensively to the substratum at the cellular periphery while SH-SY5Y cells are firmly attached by multiple protrusions. Magnification bars = 2 µm. (<b>B</b>) Inhibition of myosin contraction does not protect from SLO induced lysis. (<b>C</b>) Lysosomal exocytosis (β-hexosaminidase release) after SLO-injury is more pronounced in SH-SY5Y cells compared to HEK 293 cells. (<b>D</b>) Vacuolin-1 does not increase the SLO-induced lysis in HEK 293 cells. In contrast, Vacuolin-1-treated SH-SY5Y cells are more prone to the SLO-induced lysis. *p<0.01.</p

Self-inflicted mechanical damage in SLO-perforated cells.

<p>(<b>A–C</b>) The SLO-perforated protrusions of SH-SY5Y or HEK 293 cells are wrenched apart by mechanical forces. (<b>A</b>) The global translocation of annexin A1-YFP manifests cell lysis. (<b>B,C</b>) The cytoplasmic localization of annexin A1-YFP within the cell body is evidence of successful resealing of the plasmalemma. The arrow in (<b>B</b>) points at the resealed base of the destroyed protrusion. The asterisks in (B,<b>C</b>) denote regions of clear demarcation between permeabilized (plasmalemmal localization of annexin A1-YFP) and non-permeabilized (cytoplasmic localization of annexin A1-YFP) cellular compartments. Magnification bars = 5 µm.</p

The plasmalemmal repair of SLO pores occurs in lysosome-free cellular protrusions.

<p>(<b>A,B</b>) SLO-induced plasmalemmal translocation/cytoplasmic back-translocation of annexin A1-YFP marks the successful elimination of the SLO-pores in (<b>A</b>) a neurite of a SH-SY5Y cell or in (<b>B</b>) a cytoplasmic protrusion of a HEK 293 cell. Arrows denote the plasmalemmal translocation of annexin A1 (plasmalemmal permeabilization). Magnified images of the squared region are shown in (<b>A</b>). Magnification bars = 5 µm.</p

Destabilization of the cortical cytoskeleton enhances microvesicle release by SLO-damaged cells and potentiates their survival.

<p>(<b>A</b>) Enhanced release of microvesicles by SLO-damaged cells, which were treated with latrunculin A. (<b>B</b>) Latrunculin A protects HEK 293 cells from SLO-induced cell lysis. (<b>C</b>) Diminished release of microvesicles by SLO-damaged cells which were treated with jasplakinolide. (<b>D</b>) Treatment with jasplakinolide results in increased cell lysis. (<b>E</b>) Calpeptin reduces microvesicle release by SLO-damaged cells. (<b>F</b>) SLO induces an increased rate of lysis in calpeptin-treated cells. **p<0.001, *p<0.01. (G) Amounts of annexin A1 shed within microvesicles were analyzed by Western Blotting in culture supernatants of SLO-treated cells pre-treated with either latrunculin (Latr/SLO), jasplakinolide (Jasp/SLO), cells treated with latrunculin without SLO treatment (Latr) or cells treated with DTT only (Contr). **p<0.001.</p

Microvesicle shedding is responsible for the initial elimination of toxin pores whereas lysosomal repair mends secondary, self-inflicted mechanical injuries.

<p>(<b>A</b>) Individual experiments in which SH-SY5Y cells either repaired efficiently (low damage; 72 cells, 3 independent experiments) or suffered from extensive plasmalemmal damage (high damage; 66 cells, 3 independent experiments) were analyzed for: (<b>B</b>) amount of microvesicles released per cell, (<b>C</b>) percentage of cells that suffered from secondary, self-inflicted mechanical damage (100% = total number of cells in each individual experiment), (<b>D</b>) percentage of cells that recovered after self-inflicted mechanical damage (100% = number of mechanically-damaged cells in each individual experiment), (<b>E</b>) contribution of lysosomal repair to total repair (100% = total number of repaired cells in each individual experiment). **p<0.001, *p<0.01.</p