scholarly journals Tissue-Specific and Inducer-Specific Differential Induction of ISG56 and ISG54 in Mice

2007 ◽  
Vol 81 (16) ◽  
pp. 8656-8665 ◽  
Author(s):  
Fulvia Terenzi ◽  
Christine White ◽  
Srabani Pal ◽  
Bryan R. G. Williams ◽  
Ganes C. Sen
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Type I Interferons ◽  
Specific Cell ◽  
Type I ◽  
Mrna Levels ◽  
Tissue Specific ◽  
Interferon Stimulated Genes ◽  
Differential Pattern

ABSTRACT The interferon-stimulated genes (ISGs) ISG56 and ISG54 are strongly induced in cultured cells by type I interferons (IFNs), viruses, and double-stranded RNA (dsRNA), which activate their transcription by various signaling pathways. Here we studied the stimulus-dependent induction of both genes in vivo. dsRNA, which is generated during virus infection, induced the expression of both genes in all organs examined. Induction was not seen in STAT1-deficient mice, indicating that dsRNA-induced gene expression requires endogenous IFN. We further examined the regulation of these ISGs in several organs from mice injected with dsRNA or IFN-β. Both ISG56 and ISG54 were widely expressed and at comparable levels. However, in organs isolated from mice injected with IFN-α the expression of ISG54 was reduced and more restricted in distribution compared with the expression level and distribution of ISG56. When we began to study specific cell types, splenic B cells showed ISG54 but not ISG56 expression in response to all agonists. Finally, in livers isolated from mice infected with vesicular stomatitis virus, the expression of ISG56, but not ISG54, was induced; this difference was observed at both protein and mRNA levels. These studies have revealed unexpected complexity in IFN-stimulated gene induction in vivo. For the first time we showed that the two closely related genes are expressed in a tissue-specific and inducer-specific manner. Furthermore, our findings provide the first evidence of a differential pattern of expression of ISG54 and ISG56 genes by IFN-α and IFN-β.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Vol 116 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Erol C. Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Author(s):  
Erol Can Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

scholarly journals TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell types in vivo

2020 ◽  
Author(s):  
Yating Liu ◽  
Anthony D. Fischer ◽  
Celine L. St. Pierre ◽  
Juan F. Macias-Velasco ◽  
Heather A. Lawson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Regulation ◽  
Transcript Abundance ◽  
Cell Types ◽  
Regulatory Elements ◽  
Model Organisms ◽  
Specific Cell ◽  
Translation Efficiency ◽  
Mrna Levels

AbstractThe alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance (AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we piloted a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance (ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ∼1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.

Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells

1993 ◽  
Vol 105 (4) ◽  
pp. 1025-1043 ◽  
Author(s):  
M. Berryman ◽  
Z. Franck ◽  
A. Bretscher
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Cytoskeletal Proteins ◽  
Specific Cell ◽  
Apical Surface ◽  
Tissue Sections ◽  
Differentiated Cells

Ezrin and moesin are two cytoskeletal proteins originally purified from human placenta that are 74% identical in overall protein sequence. They are believed to be membrane-cytoskeletal linking proteins because they share sequence homology with erythrocyte band 4.1 and colocalize with actin specifically in microvilli and membrane ruffles in cultured cells. To determine if ezrin and moesin share similar distributions in vivo, we studied their localizations with respect to F-actin in tissue sections. Surprisingly, ezrin and moesin exhibited very different cellular distributions. Ezrin was highly concentrated and colocalized with actin on the apical surface of many epithelial cell types. During enterocyte differentiation, the pattern of expression and redistribution of ezrin was consistent with it performing a role in microvillus assembly. Immunoelectron microscopy in differentiated cells revealed that ezrin was restricted mainly to the plasma membrane of microvilli and other actin-rich surface projections. Moesin was found in endothelial cells and was also enriched in the apical microvilli of a restricted set of epithelial cells. All polarized cell types with abundant microvilli contained one or both proteins, suggesting that ezrin and moesin perform related functions. However, the differential expression of ezrin and moesin indicates that they have distinct properties, which are uniquely adapted to specific cell types.

Human Mesenchymal Stem Cells Differentiate Promptly into Tissue-Specific Cell Types without Cell Fusion, Mitochondrial or Membrane Vesicular Transfer in Fetal Sheep.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 436-436 ◽  
Author(s):  
Evan J. Colletti ◽  
Judith A. Airey ◽  
Esmail D. Zanjani ◽  
Christopher D. Porada ◽  
Graça Almeida-Porada
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Fusion ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Specific Cell ◽  
Type I ◽  
Fetal Sheep ◽  
Tissue Specific ◽  
The Brain

Abstract Despite the exciting reports regarding the ability of human mesenchymal stem cells (MSC) to differentiate into different cells of different organs, the mechanism by which this process occurs remains controversial. Several possible explanations have been put forth as an alternative to the existence of a true differentiation mechanism. We previously showed that MSC, at a single cell level, are able to differentiate into cells of different germ cell layers. In the present study, we investigated whether transfer of mitochondria or membrane-derived vesicles between cells and/or cell fusion participate in the events that lead to the change of phenotype of MSC upon transplantation (Tx). To this end, 54 sheep fetuses (55–60 gestational days) were Tx intra-peritoneally with Stro-1+,CD45−, Gly-A- MSC labeled prior to Tx with either CFSE, that irreversibly couples to both intracellular and cell-surface proteins, or DiD that efficiently labels all cell membranes and intracellular organelles, such as mitochondria. Evaluation of the recipients’ different organs started at 20h post-Tx and continued at 25,30,40,60 and 120h. MSC reached the liver at 25h post-Tx (0.033%±0.0) with maximal engraftment at 40h (0.13%±0.02). MSC were first detected in the lung (0.028%±0.0) and brain (0.034%±0.0) at 30h and 40h respectively. In the brain, engraftment peaked at 60 hours post-Tx (0.08%±0.0) and in the lung at 120h (0.09%±0.01). Normalization of the number of engrafted cells per tissue mass and number of Tx cells revealed that 26% of the Tx MSC reached the lung; 2% the liver; and 3% the brain. Since the decreasing number of CFSE+ and DiD+ cells detected after 120h could be due to cell division, Ki67 staining was performed and revealed that 85–95% of the engrafted cells proliferated upon lodging in the organs, and divided throughout the evaluation period. To determine MSC differentiative timeline, confocal microscopy was performed to assess whether CFSE+ or DiD+ cells expressed tissue-specific markers (MSC were negative for these markers prior to transplant) within the engrafted organs. In the liver at 25h post-Tx, all CFSE+ or DiD+ cells co-expressed alpha-fetoprotein, demonstrating the rapid switch from an MSC to a fetal hepatocyte-like phenotype. In the lung, co-localization of pro-surfactant protein and CFSE/DiD was first detected at 30h post-Tx, but cells remained negative for Caveolin1; a phenotype that is consistent with differentiation to a type II epithelial cell, but not to a more mature type I. In the brain, MSC expressed Tau promptly, but synaptophysin expression was not detected until 120h. In situ hybridization on serial sections using either a human- or sheep-specific probe, with simultaneous visualization of CFSE+ or DiD+ cells allowed us to show that no membrane or mitochondrial transfer had occurred, since none of the sheep cells contained CFSE or DiD, and all of the dye+ cells hybridized only to the human probe. Furthermore, this combined methodology enabled us to determine that differentiation to all of the different cell types had occurred in the absence of cell fusion. In conclusion, MSC engraft multiple tissues rapidly, undergo proliferation, and give rise to tissue-specific cell types in the absence of cellular fusion or the transfer of mitochondria or membrane vesicles.

scholarly journals Sustained hypoxia leads to the emergence of cells with enhanced growth, migratory, and promitogenic potentials within the distal pulmonary artery wall

2009 ◽  
Vol 297 (6) ◽  
pp. L1059-L1072 ◽  
Author(s):  
Maria G. Frid ◽  
Min Li ◽  
Meena Gnanasekharan ◽  
Danielle L. Burke ◽  
Miguel Fragoso ◽  
...  
Keyword(s):  
Pulmonary Artery ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Type I ◽  
Mrna Levels ◽  
Functional Characteristics ◽  
Cellular Mechanisms ◽  
Cell Markers ◽  
Type I Procollagen

All forms of chronic pulmonary hypertension (PH) are characterized by structural remodeling of the pulmonary artery (PA) media, a process previously attributed solely to changes in the phenotype of resident smooth muscle cells (SMC). However, recent experimental evidence in both systemic and pulmonary circulations suggests that other cell types, including circulating and local progenitors, contribute significantly to this process. The goal of this study was to determine if hypoxia-induced remodeling of distal PA (dPA) media involves the emergence of cells with phenotypic and functional characteristics distinct from those of resident dPA SMC and fibroblasts. In vivo, in contrast to the phenotypically uniform SMC composition of dPA media in control calves, the remodeled dPA media of neonatal calves with severe hypoxia-induced PH comprised cells exhibiting a distinct phenotype, including the expression of hematopoetic (CD45), leukocytic/monocytic (CD11b, CD14), progenitor (cKit), and motility-associated (S100A4) cell markers. Consistent with these in vivo observations, primary cell cultures isolated from dPA media of hypertensive calves yielded not only differentiated SMC, but also smaller, morphologically rhomboidal (thus termed here “R”) cells that transiently expressed CD11b, constitutively expressed the mesenchymal cell marker type I procollagen, expressed high mRNA levels of progenitor cell markers cKit, CD34, CD73, as well as for inflammatory mediators, IL-6 and MCP-1, and, with time in culture, gained expression of a myofibroblast marker, α-SM-actin. R cells exhibited highly augmented proliferative, migratory, invasive, and potent promitogenic capabilities, which were due, at least in part, to the production of PDGFs, SDF-1/CXCL12, and S100A4. These data suggest that the cellular mechanisms of dPA remodeling include the emergence of cells with phenotypic and functional characteristics markedly distinct from those of resident dPA cells.

scholarly journals In vivo tissue-specific chromatin profiling in Drosophila melanogaster using GFP-tagged nuclei

Genetics ◽  
2021 ◽  
Author(s):  
Juan Jauregui-Lozano ◽  
Kimaya Bakhle ◽  
Vikki M Weake
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Specific Cell ◽  
Tissue Specific ◽  
Histone Marks ◽  
Chromatin Profiling ◽  
Photoreceptor Neurons

Abstract The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.

scholarly journals Tissue specific targeting of DNA nanodevices in a multicellular living organism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kasturi Chakraborty ◽  
Palapuravan Anees ◽  
Sunaina Surana ◽  
Simona Martin ◽  
Jihad Aburas ◽  
...  
Keyword(s):  
Living Organism ◽  
Cell Types ◽  
Specific Cell ◽  
Full Potential ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Targeting ◽  
Synthetic Receptor ◽  
Ligand Interactions

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

scholarly journals A minimal RNA ligand for potent RIG-I activation in living mice

2017 ◽  
Author(s):  
Melissa M. Linehan ◽  
Thayne H. Dickey ◽  
Emanuela S. Molinari ◽  
Megan E. Fitzgerald ◽  
Olga Potapova ◽  
...  
Keyword(s):  
Immune System ◽  
Type I Interferons ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Base Pairs ◽  
Stem Loop ◽  
Interferon Stimulated Genes ◽  
Rna Sensors

AbstractWe have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor. When introduced into mice, a family of short, triphosphorylated Stem Loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense. Using RNAseq, we provide the first in-vivo genome-wide view of the expression networks that are initiated upon RIG-I activation. We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors. By contrast, poly(I:C), which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10-14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.

scholarly journals Type I IFN is siloed in endosomes

2020 ◽  
Vol 117 (30) ◽  
pp. 17510-17512 ◽  
Author(s):  
Jennie B. Altman ◽  
Justin Taft ◽  
Tim Wedeking ◽  
Conor N. Gruber ◽  
Michael Holtmannspötter ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cell Types ◽  
Specific Cell ◽  
Type I ◽  
Type I Ifn ◽  
Long Term Effects ◽  
Negative Regulators ◽  
Downstream Signaling

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular “IFN silos” persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I−induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.

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