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- Al-Sayegh, M. A., et al.
(författare)
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β-actin contributes to open chromatin for activation of the adipogenic pioneer factor CEBPA during transcriptional reprograming
- 2020
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 31:23, s. 2511-2521
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Tidskriftsartikel (refereegranskat)abstract
- Adipogenesis is regulated by a cascade of signals that drive transcriptional reprogramming in adipocytes. Here, we report that nuclear actin regulates the chromatin states that establish tissue- specific expression during adipogenesis. To study the role of beta-actin in adipocyte differentiation, we conducted RNA sequencing on wild-type and beta-actin knockout mouse embryonic fibroblasts (MEFs) after reprograming to adipocytes. We found that beta-actin depletion affects induction of several adipogenic genes during transcriptional reprograming. This impaired regulation of adipogenic genes is linked to reduced expression of the pioneer factor Cebpa and is rescued by reintroducing NLS-tagged beta-actin. ATAC-Seq in knockout MEFs revealed that actin-dependent reduction of Cebpa expression correlates with decreased chromatin accessibility and loss of chromatin association of the ATPase Brg1. This, in turn, impairs CEBPB's association with its Cebpa promoter-proximal binding site during adipogenesis. We propose a role for the nuclear beta-actin pool in maintaining open chromatin for transcriptional reprogramming during adipogenic differentiation.
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| 3. |
- Ciccarelli, Michela, et al.
(författare)
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Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding
- 2023
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 34:11
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Tidskriftsartikel (refereegranskat)abstract
- Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1 increment strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1 increment strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1 increment strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1 increment construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.
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| 4. |
- Figueroa-Cuilan, Wanda M., et al.
(författare)
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Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium
- 2023
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 34:7
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Tidskriftsartikel (refereegranskat)abstract
- Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.
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| 8. |
- Neuhaus, Mathis, et al.
(författare)
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EHD2 regulates plasma membrane integrity and downstream insulin receptor signalling events
- 2023
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Ingår i: Molecular Biology of the Cell. - 1939-4586. ; 34:12
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Tidskriftsartikel (refereegranskat)abstract
- Adipocyte dysfunction is a crucial driver of insulin resistance and type 2 diabetes. We identified EH domain-containing protein 2 (EHD2) as one of the most highly upregulated genes at the early stage of adipose tissue expansion. EHD2 is a dynamin-related ATPase influencing several cellular processes, including membrane recycling, caveolae dynamics and lipid metabolism. Here, we investigated the role of EHD2 in adipocyte insulin signalling and glucose transport. Using C57BL6/N EHD2 knockout mice under short-term high-fat diet conditions and 3T3-L1 adipocytes we demonstrate that EHD2 deficiency is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. Furthermore, we show that lack of EHD2 is linked with altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation. In conclusion, these data highlight the importance of EHD2 for the integrity of the plasma membrane milieu, insulin receptor stability, and downstream insulin receptor signalling events, involved in glucose uptake and ultimately underscore its role in insulin resistance and obesity.
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| 9. |
- Saini, P. K., et al.
(författare)
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The PSI+ prion modulates cytochrome c oxidase deficiency caused by deletion of COX12
- 2022
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 33:14
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae.cox12 strain for similar to 300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Delta cox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.
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| 10. |
- Saini, Pawan Kumar, et al.
(författare)
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The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12
- 2022
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 33:14
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.
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| 11. |
- Seal, Srijit, et al.
(författare)
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From pixels to phenotypes : Integrating image-based profiling with cell health data as BioMorph features improves interpretability
- 2024
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 35:3
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Tidskriftsartikel (refereegranskat)abstract
- Cell Painting assays generate morphological profiles that are versatile descriptors of biological systems and have been used to predict in vitro and in vivo drug effects. However, Cell Painting features extracted from classical software such as CellProfiler are based on statistical calculations and often not readily biologically interpretable. In this study, we propose a new feature space, which we call BioMorph, that maps these Cell Painting features with readouts from comprehensive Cell Health assays. We validated that the resulting BioMorph space effectively connected compounds not only with the morphological features associated with their bioactivity but with deeper insights into phenotypic characteristics and cellular processes associated with the given bioactivity. The BioMorph space revealed the mechanism of action for individual compounds, including dual-acting compounds such as emetine, an inhibitor of both protein synthesis and DNA replication. Overall, BioMorph space offers a biologically relevant way to interpret the cell morphological features derived using software such as CellProfiler and to generate hypotheses for experimental validation.
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| 12. |
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