Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.Peer reviewe

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention

Metabolomics reveals the metabolic shifts following an intervention with rye bread in postmenopausal women- a randomized control trial

<p>Abstract</p> <p>Background</p> <p>Epidemiological studies have consistently shown that whole grain (WG) cereals can protect against the development of chronic diseases, but the underlying mechanism is not fully understood. Among WG products, WG rye is considered even more potent because of its unique discrepancy in postprandial insulin and glucose responses known as the rye factor. In this study, an NMR-based metabolomics approach was applied to study the metabolic effects of WG rye as a tool to determine the beneficial effects of WG rye on human health.</p> <p>Methods</p> <p>Thirty-three postmenopausal Finnish women with elevated serum total cholesterol (5.0-8.5 mmol/L) and BMI of 20–33 kg/m² consumed a minimum of 20% of their daily energy intake as high fiber WG rye bread (RB) or refined wheat bread (WB) in a randomized, controlled, crossover design with two 8-wk intervention periods separated by an 8-wk washout period. At the end of each intervention period, fasting serum was collected for NMR-based metabolomics and the analysis of cholesterol fractions. Multilevel partial least squares discriminant analysis was used for paired comparisons of multivariate data.</p> <p>Results</p> <p>The metabolomics analysis of serum showed lower leucine and isoleucine and higher betaine and N,N-dimethylglycine levels after RB than WB intake. To further investigate the metabolic effects of RB, the serum cholesterol fractions were measured. Total- and LDL-cholesterol levels were higher after RB intake than after WB (p<0.05).</p> <p>Conclusions</p> <p>This study revealed favorable shifts in branched amino acid and single carbon metabolism and an unfavorable shift in serum cholesterol levels after RB intake in postmenopausal women, which should be considered for evaluating health beneficial effects of rye products.</p

TGF-β-Elicited Induction of Tissue Inhibitor of Metalloproteinases (TIMP)-3 Expression in Fibroblasts Involves Complex Interplay between Smad3, p38α, and ERK1/2

Transforming growth factor-β (TGF-β) promotes extracellular matrix deposition by down-regulating the expression of matrix degrading proteinases and upregulating their inhibitors. Tissue inhibitor of metalloproteinases (TIMP)-3 is an ECM-associated specific inhibitor of matrix degrading metalloproteinases. Here, we have characterized the signaling pathways mediating TGF-β-induced expression of TIMP-3. Basal and TGF-β-induced TIMP-3 mRNA expression was abolished in Smad4-deficient mouse embryonic fibroblasts and restoring Smad4 expression rescued the response. Inhibition of Smad signaling by expression of Smad7 and dominant negative Smad3 completely abolished TGF-β-elicited expression of TIMP-3 in human fibroblasts, whereas overexpression of Smad3 enhanced it. Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression. Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression. These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis

Genetic architecture of human plasma lipidome and its link to cardiovascular disease

Abstract Understanding genetic architecture of plasma lipidome could provide better insights into lipid metabolism and its link to cardiovascular diseases (CVDs). Here, we perform genome-wide association analyses of 141 lipid species (n = 2,181 individuals), followed by phenome-wide scans with 25 CVD related phenotypes (n = 511,700 individuals). We identify 35 lipid-species-associated loci (P &lt;5 ×10−8), 10 of which associate with CVD risk including five new loci-COL5A1, GLTPD2, SPTLC3, MBOAT7 and GALNT16 (false discovery rate&lt;0.05). We identify loci for lipid species that are shown to predict CVD e.g., SPTLC3 for CER(d18:1/24:1). We show that lipoprotein lipase (LPL) may more efficiently hydrolyze medium length triacylglycerides (TAGs) than others. Polyunsaturated lipids have highest heritability and genetic correlations, suggesting considerable genetic regulation at fatty acids levels. We find low genetic correlations between traditional lipids and lipid species. Our results show that lipidomic profiles capture information beyond traditional lipids and identify genetic variants modifying lipid levels and risk of CVD