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Ca²⁺-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

ABSTRACT: The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fc? receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex beads, Escherichia coli, Salmonella typhimurium, and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3, and dynamin-2. Brefeldin A treatment inhibited mannosidase-II recruitment and phagocytic uptake of serum-coated or -uncoated latex beads and E. coli However, consistent with previous studies, brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically, recruitment of mannosidase-II vesicles during phagocytic uptake required Ca²⁺ from both extra- and intracellular sources apart from PI3K, microtubules, and dynamin-2. Extracellular Ca²⁺ via voltage-gated Ca²⁺ channels established a Ca²⁺-dependent local phosphatidylinositol 1,4,5-trisphosphate gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1, whereas recruitment of the recycling endosome marker VAMP3 was unaffected. Depletion of both VAMP2 and NCS1 individually resulted in the reduced uptake by macrophages. Together, the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function.

SUBMITTER: Vashi N

PROVIDER: S-EPMC5392664 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Ca<sup>2+</sup>-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

Vashi Nimi N Andrabi Syed Bilal Ahmad SB Ghanwat Swapnil S Suar Mrutyunjay M Kumar Dhiraj D

The Journal of biological chemistry 20170207 13

The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fcγ receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex ...[more]

PMID: 28174296

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Project description:BackgroundRadiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms.MethodsEndothelial-specific MCU (mitochondrial Ca2+ uniporter) knockout and C57Bl6/J mice with or without a preinfusion of a mitoTEMPO (mitochondrial reactive oxygen species [ROS] scavenger) were exposed to a single dose of cranial irradiation. 24, and 240 hours postirradiation, vascular reactivity, endothelial function, and mitochondrial integrity were assessed ex vivo and in vitro.ResultsIn cultured human endothelial cells, irradiation with 4 Gy increased cytosolic Ca2+ transients and the mitochondrial Ca2+ concentration ([Ca2+]mt) and activated MCU. These outcomes correlated with increases in mitochondrial ROS (mtROS), loss of NO production, and sustained damage to mitochondrial but not nuclear DNA. Moreover, irradiation impaired activity of the ETC (electron transport chain) and the transcription of ETC subunits encoded by mitochondrial DNA (mtDNA). Knockdown or pharmacological inhibition of MCU blocked irradiation-induced mtROS production, mtDNA damage, loss of NO production, and impairment of ETC activity. Similarly, the pretreatment with mitoTEMPO, a scavenger of mtROS, reduced irradiation-induced Ca2+ entry, and preserved both the integrity of the mtDNA and the production of NO, suggesting a feed-forward loop involving [Ca2+]m and mtROS. Enhancement of DNA repair in mitochondria, but not in the nucleus, was sufficient to block prolonged mtROS elevations and maintain NO production. Consistent with the findings from cultured cells, in C57BL/6J mice, head and neck irradiation decreased endothelium-dependent vasodilation, and mtDNA integrity in the carotid artery after irradiation. These effects were prevented by endothelial knockout of MCU or infusion with mitoTEMPO.ConclusionsIrradiation-induced damage to mtDNA is driven by MCU-dependent Ca2+ influx and the generation of mtROS. Such damage leads to reduced transcription of mitochondrial genes and activity of the ETC, promoting sustained mtROS production that induces endothelial dysfunction. Our findings suggest that targeting MCU and mtROS might be sufficient to mitigate irradiation-induced vascular disease.

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Ca²⁺-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

Publications

Ca<sup>2+</sup>-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

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Dataset Information

Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

Publications

Ca<sup>2+</sup>-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Ca²⁺-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.