11 research outputs found
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Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells
The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells
Mitochondria in precision medicine; linking bioenergetics and metabolomics in platelets
Mitochondria possess reserve bioenergetic capacity, supporting protection and resilience in the face of disease. Approaches are limited to understand factors that impact mitochondrial functional reserve in humans. We applied the mitochondrial stress test (MST) to platelets from healthy subjects and found correlations between energetic parameters and mitochondrial function. These parameters were not correlated with mitochondrial complex I-IV activities, however, suggesting that other factors affect mitochondrial bioenergetics and metabolism. Platelets from African American patients with sickle cell disease also differed from controls, further showing that other factors impact mitochondrial bioenergetics and metabolism. To test for correlations of platelet metabolites with energetic parameters, we performed an integrated analysis of metabolomics and MST parameters. Subsets of metabolites, including fatty acids and xenobiotics correlated with mitochondrial parameters. The results establish platelets as a platform to integrate bioenergetics and metabolism for analysis of mitochondrial function in precision medicine
Ribosome-associated Vesicles: A Dynamic Subcompartment of the Endoplasmic Reticulum in Secretory Cells
The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.Support for this study was provided by the L. V. Gerstner Jr., Scholars Program (to Z.F.), the Leon Levy Foundation (to Z.F.), the John F. and Nancy A. Emmerling Fund of the Pittsburgh Foundation (to Z.F.), the Department of Defense PR141292 (to Z.F.), NIH K08DA031241 (to Z.F.), NSF MCB-1408986 (to S.A.M.), the National Science Foundation Graduate Research Fellowship (to N.H.T.), NIH K01AG045335 (to E.A.G.), NIH 1S10RR019003 (to S.C.W.), NIH 1S10RR025488 (to S.C.W.), NIH 1S10RR016236 (to S.C.W.), NIH F30NS093798 (to S.E.S.), NIH R56AG058593 (to Z.P.W.), the Howard Hughes Medical Institute (to P.W., N.H.T., J.F., and G.J.J.), NIH GM29169 (to J.F.), NIH GM122588 (to G.J.J.), NIH AI150464 (to G.J.J.), the Israel Science Foundation Grant 1285/14 (to S.G.W.), the European Research Council under the European Union’s Seventh Framework Programme (grant number 310649) (to D.F.), MINECO AIC-A-2011-0638 (to J.M.C.), the Spanish Ministry of Economy and Competitiveness grant BIO2016-76400-R AEI/FEDER, UE (to J.M.C.),
and Comunidad Autónoma de Madrid grant S2017/BMD-3817 (to J.M.C.). Some of the cryo-ET was performed in the Beckman Institute Resource Center for Transmission EM at Caltech. Additional work was also performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310) with added support from NIH S10 RR029300-01. CSTET data acquisition was partially supported by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science. Some of the live confocal images were collected and processed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University and supported by NIH P30 CA013696. Part of the cryo-EM image processing was conducted as an Instruct-ERIC collaboration project PD1222 at the Instruct Image Processing CenterPeer reviewe
BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension
International audienc
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BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension
BackgroundDeficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined.MethodsIn vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH.ResultsIn cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation.ConclusionsBOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development