scholarly journals Cells recognize osmotic stress through liquid-liquid phase separation lubricated with poly(ADP-ribose)

2020 ◽  
Author(s):  
Kengo Watanabe ◽  
Kazuhiro Morishita ◽  
Xiangyu Zhou ◽  
Shigeru Shiizaki ◽  
Yasuo Uchiyama ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Osmotic Stress ◽  
Cell Volume ◽  
Hyperosmotic Stress ◽  
Cell Swelling ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Genome Wide ◽  
A Genome ◽  
Interfering Rna

AbstractCells are under threat of osmotic perturbation; and cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we report that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identified an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems.

scholarly journals Cells recognize osmotic stress through liquid–liquid phase separation lubricated with poly(ADP-ribose)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kengo Watanabe ◽  
Kazuhiro Morishita ◽  
Xiangyu Zhou ◽  
Shigeru Shiizaki ◽  
Yasuo Uchiyama ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Osmotic Stress ◽  
Cell Volume ◽  
Hyperosmotic Stress ◽  
Cell Swelling ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Genome Wide ◽  
A Genome ◽  
Interfering Rna

AbstractCells are under threat of osmotic perturbation; cell volume maintenance is critical in cerebral edema, inflammation and aging, in which prominent changes in intracellular or extracellular osmolality emerge. After osmotic stress-enforced cell swelling or shrinkage, the cells regulate intracellular osmolality to recover their volume. However, the mechanisms recognizing osmotic stress remain obscured. We previously clarified that apoptosis signal-regulating kinase 3 (ASK3) bidirectionally responds to osmotic stress and regulates cell volume recovery. Here, we show that macromolecular crowding induces liquid-demixing condensates of ASK3 under hyperosmotic stress, which transduce osmosensing signal into ASK3 inactivation. A genome-wide small interfering RNA (siRNA) screen identifies an ASK3 inactivation regulator, nicotinamide phosphoribosyltransferase (NAMPT), related to poly(ADP-ribose) signaling. Furthermore, we clarify that poly(ADP-ribose) keeps ASK3 condensates in the liquid phase and enables ASK3 to become inactivated under hyperosmotic stress. Our findings demonstrate that cells rationally incorporate physicochemical phase separation into their osmosensing systems.

scholarly journals Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

2017 ◽  
Vol 114 (40) ◽  
pp. E8362-E8371 ◽  
Author(s):  
Anna Vilborg ◽  
Niv Sabath ◽  
Yuval Wiesel ◽  
Jenny Nathans ◽  
Flonia Levy-Adam ◽  
...  
Keyword(s):  
Heat Shock ◽  
Osmotic Stress ◽  
Stress Responses ◽  
Failure Process ◽  
Mouse Fibroblast ◽  
Open Chromatin ◽  
Chromatin Signature ◽  
Genomic Features ◽  
Genome Wide ◽  
A Genome

Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.

Effects of osmotic stress on the activity of MAPKs and PDGFR-β-mediated signal transduction in NIH-3T3 fibroblasts

2008 ◽  
Vol 294 (4) ◽  
pp. C1046-C1055 ◽  
Author(s):  
M.-B. Nielsen ◽  
S. T. Christensen ◽  
E. K. Hoffmann
Keyword(s):  
Signal Transduction ◽  
Osmotic Stress ◽  
Cell Volume ◽  
Nuclear Translocation ◽  
Cell Swelling ◽  
Cell Shrinkage ◽  
Jnk Pathway ◽  
Mapk Activity ◽  
3T3 Fibroblasts ◽  
Nih 3T3

Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-β-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-β and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-β, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-β activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-β and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.

A genome-wide survey of small interfering RNA and microRNA pathway genes in a galling insect

2013 ◽  
Vol 59 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Jacob T. Shreve ◽  
Richard H. Shukle ◽  
Subhashree Subramanyam ◽  
Alisha J. Johnson ◽  
Brandon J. Schemerhorn ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Genome Wide ◽  
A Genome ◽  
Microrna Pathway ◽  
Interfering Rna ◽  
Genome Wide Survey

scholarly journals Resting and osmotically induced basolateral K conductances in turtle colon.

1986 ◽  
Vol 88 (2) ◽  
pp. 253-274 ◽  
Author(s):  
W J Germann ◽  
S A Ernst ◽  
D C Dawson
Keyword(s):  
Epithelial Cells ◽  
Osmotic Stress ◽  
Cell Volume ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Na Transport ◽  
Colonic Epithelial Cells ◽  
Cell Layers ◽  
Normal Transport

Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCl) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling. In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidine-sensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a new conductance, which was highly selective for K over Rb and was blocked by quinidine or lidocaine.

scholarly journals Genome-wide identification of endothelial cell–enriched genes in the mouse embryo

Blood ◽  
2012 ◽  
Vol 120 (4) ◽  
pp. 914-923 ◽  
Author(s):  
Haruka Takase ◽  
Ken Matsumoto ◽  
Rie Yamadera ◽  
Yoshiaki Kubota ◽  
Ayaka Otsu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Vascular Development ◽  
Tube Formation ◽  
Vascular Plexus ◽  
Genome Wide ◽  
A Genome ◽  
Human Orthologs ◽  
Rna Knockdown ◽  
Interfering Rna

Abstract The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo–forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.

Effects of P-glycoprotein on cell volume regulation in mouse proximal tubule

2001 ◽  
Vol 280 (5) ◽  
pp. F829-F837 ◽  
Author(s):  
Yukio Miyata ◽  
Yasushi Asano ◽  
Shigeaki Muto
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Volume Regulation ◽  
Control Volume ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Hyperosmotic Stress ◽  
Cell Swelling ◽  
Bathing Solution ◽  
P Glycoprotein

The role of P-glycoprotein (P-gp) in cell volume regulation was examined in isolated nonperfused proximal tubule S2 segments from wild-type (WT) mice and those in which both mdr1a and mdr1b genes were knocked out (KO). When the osmolality of the bathing solution was rapidly decreased from 300 to 180 mosmol/kgH2O, the tubules from both the WT and KO mice exhibited regulatory volume decrease (RVD) by a similar magnitude after the initial cell swelling. The peritubular addition of two P-pg inhibitors (verapamil and cyclosporin A) to either group of the tubules had no effect on RVD. When the tubules from the WT mice were rapidly exposed to a hyperosmotic solution (500 mosmol/kgH2O) including 200 mM mannitol, they abruptly shrank to 82.1% of their control volume but remained in a shrunken state during the experimental period, indicating a lack of regulatory volume increase (RVI). The addition of the two P-gp inhibitors, but not the inhibitor of the renal organic cation transport system (tetraethylammonium), to the tubules from the WT mice resulted in RVI. Surprisingly, when the tubules from the KO mice were exposed to the hyperosmotic solution, they abruptly shrank to 79.9% of their control volume, and then gradually swelled to 87.7% of their control volume, showing RVI. However, exposure of the tubules from the KO mice to the hyperosmotic solution in the presence of the two P-gp inhibitors had no effect on RVI. When the tubules of the WT mice were exposed to the hyperosmotic solution including either of the two P-gp inhibitors, in the absence of peritubular Na+ or in the presence of peritubular ethylisopropylamiloride (EIPA; the specific inhibitor of Na+/H+ exchange), they did not exhibit RVI. In the tubules of the KO mice, both removing peritubular Na+and adding peritubular EIPA inhibited RVI induced by the hyperosmotic solution. We conclude that 1) in mouse proximal tubule, P-gp modulates RVI during hyperosmotic stress but not RVD during hyposmotic stress and 2) basolateral membrane Na+/H+ exchange partly contributes to the P-gp-induced modulation of RVI under hyperosmotic stress.

scholarly journals Lysosomal degradation products induceCoxiella burnetiivirulence

2020 ◽  
Vol 117 (12) ◽  
pp. 6801-6810 ◽  
Author(s):  
Patrice Newton ◽  
David R. Thomas ◽  
Shawna C. O. Reed ◽  
Nicole Lau ◽  
Bangyan Xu ◽  
...  
Keyword(s):  
Degradation Products ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Genome Wide ◽  
Multiple Protein ◽  
A Genome ◽  
Genes Encoding ◽  
Reporter Strains ◽  
Type Ivb Secretion ◽  
Interfering Rna

Coxiella burnetiiis an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of theC. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical forC. burnetiivirulence. Indeed, theC. burnetiiPmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate theC. burnetiiPmrA/B two-component system. This study has further enhanced our understanding ofC. burnetiipathogenesis, the host–pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.

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