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Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change [ChIP-seq]

ABSTRACT: Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membrane-less compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ~10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ~100 s) with minimal impact on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Together, our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration that adapts the cell to volume change.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156227 | GEO | 2020/08/14

REPOSITORIES: GEO

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Project description:A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4M-BM-0C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses Control cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl stress which had three biological replicates) were labeled with Cy3 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Sample cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl which had three biological replicates) were labeled with Cy5 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Condition one M-bM-^@M-^Ssub-lethal hyperosmotic stress: Control culture (Cy3-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) M-bM-^@M-^S strains grown to mid exponential growth at 25M-BM-0C phase (in a shaking water bath) in BHI broth supplemented with 8% additional NaCl (strain 701700), 10% additional NaCl (strain ATCC19115) or 12% additional NaCl (ScottA). Condition two M-bM-^@M-^S cold stress: Control culture (Cy3-labelled RNA) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA) M-bM-^@M-^S strains grown to exponential growth phase at 4M-BM-0C in BHI broth.

Project description:Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using E. coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ~1.0 Os kg-1 causes a dose-dependent K+ leak from the cell, resulting in a substantial decrease in cytosolic K+ content and a concurrent accumulation of Na+ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K+ uptake. Ion flux data is consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K+ uptake to efflux. Microarray experiments reveal that about 40% of up-regulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic- and non-ionic osmotica The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K+ transporters for bacterial adaptation to hyperosmotic stress. Experiment Overall Design: Two biological replicates per treatment with microarray analysis using the Affymetrix GeneChip E. coli Genome array. Treatments used included: Experiment Overall Design: Control - E. coli Frag1, grown to early stationary growth phase in a mineral salts medium with 0.1% glucose at 25 C Experiment Overall Design: Sucrose hyperosmotic treatment - 1.25 M sucrose added to control culture Experiment Overall Design: for 10 min. Experiment Overall Design: NaCl hyperosmotic treatment - 1.37 M NaCl added to control culture Experiment Overall Design: For microarray data comparisons the sucrose and NaCl hyperosmotic treatment data was compared to the no treatment control data separately. The sucrose to control and NaCL to control comparison data tables are linked below.

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Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change [ChIP-seq]

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