Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Microbial tolerance in secondary peritonitis is dose dependent

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48 h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48 h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24 h (median: 5.04 log CFU) and 48 h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48 h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10⁵ CFU and 2 × 10⁸ CFU Klebsiella. Cells harvested from animals 48 h after infection, were unresponsive to an ex vivo stimulation with 10⁵ CFU Klebsiella, but fully responded to 10⁸ CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.     

CYTOLOGY OF DIFFERENTIATING TRACHEARY ELEMENTS I. ORGANELLES AND MEMBRANE SYSTEMS

The distribution of organelles, membrane systems, and ribosomes is not at any time obviously related to the pattern of secondary wall in helically thickened tracheary elements in leaves of Beta vulgaris L. (sugar beet) and Cucurbita maxima Duchesne, fixed with potassium permanganate and osmium tetroxide. During the differentiation of the secondary wall, cisternae of the endoplasmic reticulum and dictyosomes are particularly conspicuous, and the dictyosomes are associated with numerous vesicles. Similar vesicles appear to be in various stages of fusion with the secondary wall thickenings. The tracheary elements contain plastids which may include starch granules. Ribosomes occur free in the cytoplasm and in association with endoplasmic membranes.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.