bims-nenemi 2023-04-16 papers

Issue of 2023‒04‒16
seven papers selected by
Marco Tigano
Thomas Jefferson University

Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2210623120

A genome-wide optical pooled screen reveals regulators of cellular antiviral responses.

Rebecca J Carlson, Michael D Leiken, Alina Guna, Nir Hacohen, Paul C Blainey.

The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.

Keywords: CRISPR screening; IRF3; RIG-I; Sendai; high-content imaging

DOI: https://doi.org/10.1073/pnas.2210623120

Int J Mol Sci. 2023 Mar 24. pii: 6128. [Epub ahead of print]24(7):

Mitochondrial Signaling Pathways Associated with DNA Damage Responses.

Tsutomu Shimura.

Under physiological and stress conditions, mitochondria act as a signaling platform to initiate biological events, establishing communication from the mitochondria to the rest of the cell. Mitochondrial adenosine triphosphate (ATP), reactive oxygen species, cytochrome C, and damage-associated molecular patterns act as messengers in metabolism, oxidative stress response, bystander response, apoptosis, cellular senescence, and inflammation response. In this review paper, the mitochondrial signaling in response to DNA damage was summarized. Mitochondrial clearance via fusion, fission, and mitophagy regulates mitochondrial quality control under oxidative stress conditions. On the other hand, damaged mitochondria release their contents into the cytoplasm and then mediate various signaling pathways. The role of mitochondrial dysfunction in radiation carcinogenesis was discussed, and the recent findings on radiation-induced mitochondrial signaling and radioprotective agents that targeted mitochondria were presented. The analysis of the mitochondrial radiation effect, as hypothesized, is critical in assessing radiation risks to human health.

Keywords: DNA damage response; inflammation response; mitochondrial signaling; oxidative stress; radiation carcinogenesis

DOI: https://doi.org/10.3390/ijms24076128

Immunity. 2023 Apr 11. pii: S1074-7613(23)00132-2. [Epub ahead of print]56(4): 742-744

An orphan no more: Nur77 senses cytosolic LPS.

Megan H Orzalli.

Cytosolic LPS activates the NLRP3 inflammasome via a gasdermin D (GSDMD)-dependent mechanism. In this issue of Immunity, Zhu et al.1 provide insight into the events linking these two steps, identifying the orphan nuclear receptor Nur77 as a mediator of NLRP3 activation that senses LPS and GSDMD-dependent accumulation of cytosolic mtDNA.

DOI: https://doi.org/10.1016/j.immuni.2023.03.012

Sci Rep. 2023 Apr 08. 13(1): 5788

Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells.

Vojtěch Pavluch, Tomáš Špaček, Hana Engstová, Andrea Dlasková, Petr Ježek.

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25-40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.

DOI: https://doi.org/10.1038/s41598-023-33012-6

Cells. 2023 Apr 05. pii: 1089. [Epub ahead of print]12(7):

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Keywords: NSCs; TMRM; co-culture; machine learning; mitochondrial membrane potential; single muscle fibers; spheroids

DOI: https://doi.org/10.3390/cells12071089

J Virol Methods. 2023 Apr 12. pii: S0166-0934(23)00056-3. [Epub ahead of print] 114731

Generation of an A549 ISRE-Luciferase Stable Cell Line.

Laurie-Anne Lamotte, Lionel Tafforeau.

With its human lung origin, A549 cell line is a designated cellular model for viral respiratory infections studies. As such infections are known to lead to innate immune responses, various IFN signaling modifications occur in infected cells and have to be considered in respiratory viruses experiments. Here, we describe the generation of an A549 stable cell line that expresses firefly luciferase upon interferon-β stimulation, as well as upon RIG-I transfection and upon influenza A virus infection. Of the 18 clones generated, the first one, namely A549-RING1, demonstrated appropriate luciferase expression in the different conditions tested. This newly established cell line may therefore be used to decipher the impact of viral respiratory infection on innate immune response depending on IFN stimulation, without any plasmid transfection step. A549-RING1 can be provided upon request.

Keywords: IFN-β; ISRE-Luciferase; Influenza A virus; RIG-I; innate immune response; viral infection

DOI: https://doi.org/10.1016/j.jviromet.2023.114731

Ecotoxicol Environ Saf. 2023 Apr 10. pii: S0147-6513(23)00401-3. [Epub ahead of print]256 114897

Ambient particulate matter exposure plus chronic ethanol ingestion exacerbates hepatic fibrosis by triggering the mitochondrial ROS-ferroptosis signaling pathway in mice.

Shibin Ding, Jinjin Jiang, Guofu Zhang, Min Yu, Yang Zheng.

BACKGROUND: Chronic ethanol ingestion causes persistent oxidative stresses in the liver, leading to hepatic injury and fibrosis, but the underlying mechanisms remain unclear. Recently, ambient particulate matter (PM) has been confirmed to aggravate high-fat diet-induced liver fibrosis by enhancing oxidative stress. Thus, we hypothesized that oxidative stress induced by ambient PM exposure increases the severity of liver fibrosis caused by ethanol ingestion.METHODS AND RESULTS: C57BL/6 mice were subjected to ambient PM inhalation, ethanol ingestion or ambient PM-plus-ethanol ingestion for 12 weeks. Oxidative stress, mitochondrial reactive oxygen species (MtROS), liver fibrosis and ferroptosis indicators in the liver were evaluated. In vitro, oxidative stress, MtROS, ferroptosis indicators, profibrotic molecules and fibrosis markers in hepatic stellate (LX-2) cells were also determined. We found that ethanol ingestion markedly elevated hepatic oxidative stress and MtROS levels, triggered hepatic ferroptosis, and induced liver fibrosis, along with upregulation of the profibrotic molecule TGF-β1 and fibrosis marker collagen-I, in mice. Moreover, the combination of ambient PM and ethanol accelerated these adverse effects. Importantly, the combination of PM exposure and ethanol ingestion had a synergistic effect on these changes. In vitro, LX-2 cells activated with PM2.5 alone or combined with ethanol showed upregulation of TGF-β1 and collagen-I. In addition, the levels of MtROS, the oxidative stress marker 4-hydroxynonenal (4-HNE) and ferroptosis-related proteins and the GSH/GSSG ratio were significantly increased in PM2.5 plus ethanol-treated LX-2 cells. After pretreatment with a MtROS scavenger (Mito-TEMPO), we found that Mito-TEMPO treatment inhibited ferroptosis and oxidative stress in PM2.5 plus ethanol-treated LX-2 cells. Furthermore, a speciﬁc ferroptosis inhibitor (Fer-1) decreased the levels of ferroptosis-related proteins and profibrotic molecules in activated LX-2 cells co-exposed to PM2.5 and ethanol.
CONCLUSION: In this study, we revealed that ambient PM exposure induced profibrotic effects and that combined exposure to ambient PM and chronic ethanol ingestion exacerbated hepatic ﬁbrosis, which may trigger ferroptosis by increasing MtROS, thereby activating hepatic stellate cells.

Keywords: Ethanol; Ferroptosis; Hepatic fibrosis; Mitochondrial reactive oxygen species; Oxidative stress; Particulate matter

DOI: https://doi.org/10.1016/j.ecoenv.2023.114897