STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon β expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase–dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system

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An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM

Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.     

Biomediated synthesis of silver nanoparticles using Exiguobacterium mexicanum

Biomediated silver nanoparticle were synthesized using a cell free extract of a soil bacterium, Exiguobacterium mexicanum PR 10.6. The silver nanoparticles were characterised using UV–Vis spectroscopy, energy dispersive spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. The nanoparticles ranged from 5 to 40 nm. Extracellular polymeric substance played a critical role in the reduction of silver ion and nanoparticle stabilisation when using the cell free extract. The synthesis using E. mexicanum is an effective eco-friendly, rapid method for silver nanoparticle synthesis within 1 h.     

Screening for inhibitory activities of essential oils on the growth of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., the causal agent of leaf spot disease of Murraya koenigii L.

Seven essential oils namely clove, cedar wood, lemongrass, peppermint, eucalyptus, citronella and neem oils were tested for their inhibitory effect on spore germination, growth of germ tube and mycelial growth of Colletotrichum gloeosporioides isolated from diseased Murraya koenigii. All essential oils inhibited the germination and growth of germ tube at different concentrations. However, significant reduction in colony growth was observed with citrus, lemongrass and peppermint oils at 1000, 1500 and 2000 ppm concentrations, respectively. Citrus oil at 1360 ppm inhibited the maximum growth of the fungus followed by lemongrass oil at 1720 ppm and peppermint at 2260 ppm, respectively. The effect of essential oils on mycelial dry weight also showed antifungal activity on the growth of Colletotrichum gloeosporioides. The study revealed the possible utilisation of these essential oils for foliar spray for the management of leaf spot disease of Murraya koenigii.     

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes

Background

Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes.

Methodology/Principal Findings

CD62P⁺ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14⁺CD16⁻, CD14^highCD16⁺and CD14^lowCD16⁺. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14^highCD16⁺ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P⁺-platelets (r² = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14⁺CD16⁻ cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14⁺CD16⁺ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r² = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809.

Conclusions/Significance

These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14^highCD16⁺ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.