MK2-Dependent p38b Signalling Protects Drosophila Hindgut Enterocytes against JNK-Induced Apoptosis under Chronic Stress

The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress

Multiple signaling kinases target Mrc1 to prevent genomic instability triggered by transcription-replication conflicts

Conflicts between replication and transcription machineries represent a major source of genomic instability and cells have evolved strategies to prevent such conflicts. However, little is known regarding how cells cope with sudden increases of transcription while replicating. Here, we report the existence of a general mechanism for the protection of genomic integrity upon transcriptional outbursts in S phase that is mediated by Mrc1. The N-terminal phosphorylation of Mrc1 blocked replication and prevented transcription-associated recombination (TAR) and genomic instability during stress-induced gene expression in S phase. An unbiased kinome screening identified several kinases that phosphorylate Mrc1 at the N terminus upon different environmental stresses. Mrc1 function was not restricted to environmental cues but was also required when unscheduled transcription was triggered by low fitness states such as genomic instability or slow growth. Our data indicate that Mrc1 integrates multiple signals, thereby defining a general safeguard mechanism to protect genomic integrity upon transcriptional outbursts.España, MINECO BFU2015-64437-PFEDER BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2014-52333-P and FEDER to E.N.; and BFU2013-4291

Nutrient restriction enhances the proliferative potential of cells lacking the tumor suppressor PTEN in mitotic tissues

How single cells in a mitotic tissue progressively acquire hallmarks of cancer is poorly understood. We exploited mitotic recombination in developing Drosophila imaginal tissues to analyze the behavior of cells devoid of the tumor suppressor PTEN, a negative regulator of PI3K signaling, under varying nutritional conditions. Cells lacking PTEN strongly overproliferated specifically in nutrient restricted larvae. Although the PTEN mutant cells were sensitive to starvation, they successfully competed with neighboring cells by autonomous and non-autonomous mechanisms distinct from cell competition. The overgrowth was strictly dependent on the activity of the downstream components Akt/PKB and TORC1, and a reduction in amino acid uptake by reducing the levels of the amino acid transporter Slimfast caused clones of PTEN mutant cells to collapse. Our findings demonstrate how limiting nutritional conditions impact on cells lacking the tumor suppressor PTEN to cause hyperplastic overgrowth.ISSN:2050-084

Very high cycle fatigue assessment at elevated temperature of 100 μm thin structures made of high-strength steel X5CrNiCuNb16-4

Many components and structures are exposed to very high number of cycles and challenging environmental conditions during operation. This study contributes to a better understanding of the very high cycle fatigue (VHCF) properties of high-strength steel X5CrNiCuNb16-4 at room temperature (RT) and 350 °C. For this purpose, conventional specimens and thin-walled structures are extensively examined with novel high-frequency fatigue testing techniques at elevated temperature. Tests with unnotched specimens at 350 °C show a 21.7% reduction in fatigue strength for 107 cycles and a different failure mechanism compared to RT. In contrast, no temperature influence is observed for mildly notched specimens and even a higher local fatigue strength is found for sharply notched specimens at 350 °C. The decrease in fatigue strength for 109 cycles is more pronounced at 350 °C (−10%) than at RT (−5%), and it is proven that notched specimens adequately represent the VHCF behaviour of structures. The transferability of specimen results to components and structures is given great attention. A new proposal for the VHCF strength assessment of structures with high stress gradients is presented, which is based on specimen results, an extended material-mechanical support factor and a VHCF reduction factor. The prediction model gives conservative fatigue strength estimates for 109 cycles with a maximum deviation of 5.8%. This demonstrates that even complex shaped structures can be successfully evaluated with suitable specimens and methods

Fatigue life assessment regarding different influences on the HCF/VHCF behavior of a martensitic steel

Modern applications require a special treatment when the conventional specimen size is much larger than the component size. Additional to that, high sophisticated materials are used for highly loaded components. Often the conventional fatigue limit is exceeded and loads are applied in the VHCF regime. Focus was put on the lifetime calculation and the implementation of investigated fatigue data of a X5CrNiCuNb-16-4 type steel. Two specimen geometries with diameters D7.5=7.5 mm and D2.5=2.5 mm were tested at R=-1, at room temperature and up to 109 cycles to failure. The application of different software tools (FEMFAT, fe-safe) showed several issues based on the current results. Results showed a change of crack initiation mechanism to subsurface crack initiation at approx. 2x106 cycles to failure. The gradient based correction of the reference fatigue data showed a good applicability up to 2x106 cylces. The application of fe-safe allows the flexible modification of S/N parameters over the whole cycle range. The usage of the actual material configuration introduced several important questions regarding the fatigue data and the implementation into lifetime calculation tools

Fatigue life assessment regarding different influences on the HCF/VHCF behavior of a martensitic steel

Modern applications require a special treatment when the conventional specimen size is much larger than the component size. Additional to that, high sophisticated materials are used for highly loaded components. Often the conventional fatigue limit is exceeded and loads are applied in the VHCF regime. Focus was put on the lifetime calculation and the implementation of investigated fatigue data of a X5CrNiCuNb-16-4 type steel. Two specimen geometries with diameters D7.5=7.5 mm and D2.5=2.5 mm were tested at R=-1, at room temperature and up to 109 cycles to failure. The application of different software tools (FEMFAT, fe-safe) showed several issues based on the current results. Results showed a change of crack initiation mechanism to subsurface crack initiation at approx. 2x106 cycles to failure. The gradient based correction of the reference fatigue data showed a good applicability up to 2x106 cylces. The application of fe-safe allows the flexible modification of S/N parameters over the whole cycle range. The usage of the actual material configuration introduced several important questions regarding the fatigue data and the implementation into lifetime calculation tools

Untargeted metabolomics unravels functionalities of phosphorylation sites in Saccharomyces cerevisiae

Background: Coordinated through a complex network of kinases and phosphatases, protein phosphorylation regulates essentially all cellular processes in eukaryotes. Recent advances in proteomics enable detection of thousands of phosphorylation sites (phosphosites) in single experiments. However, functionality of the vast majority of these sites remains unclear and we lack suitable approaches to evaluate functional relevance at a pace that matches their detection. Results: Here, we assess functionality of 26 phosphosites by introducing phosphodeletion and phosphomimic mutations in 25 metabolic enzymes and regulators from the TOR and HOG signaling pathway in Saccharomyces cerevisiae by phenotypic analysis and untargeted metabolomics. We show that metabolomics largely outperforms growth analysis and recovers 10 out of the 13 previously characterized phosphosites and suggests functionality for several novel sites, including S79 on the TOR regulatory protein Tip41. We analyze metabolic profiles to identify consequences underlying regulatory phosphorylation events and detecting glycerol metabolism to have a so far unknown influence on arginine metabolism via phosphoregulation of the glycerol dehydrogenases. Further, we also find S508 in the MAPKK Pbs2 as a potential link for cross-talking between HOG signaling and the cell wall integrity pathway. Conclusions: We demonstrate that metabolic profiles can be exploited for gaining insight into regulatory consequences and biological roles of phosphosites. Altogether, untargeted metabolomics is a fast, sensitive and informative approach appropriate for future large-scale functional analyses of phosphosites.This work was funded through the SystemsX.ch project SignalX, evaluated by the Swiss National Science Foundation to US and ZRN and grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64437-P and FEDER), the Catalan Government (2014 SGR 599) and Fundación Botín, by Banco Santander through its Santander Universities Global Division to FP. FP is recipients of an ICREA Acadèmia (Generalitat de Catalunya). GS was supported by an Advanced Postdoc.Mobility fellowship (P300P3_147895) by the Swiss National Science Foundation

Additional file 8: of Untargeted metabolomics unravels functionalities of phosphorylation sites in Saccharomyces cerevisiae

Corrected p-values from untargeted metabolomics. (XLSX 1258 kb

The Hog1 stress-activated protein kinase targets nucleoporins to control mRNA export upon stress

15 páginas, 11 figuras. El fichero contiene una rectificación en la página 17The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPKThis work was supported by MINECO (Spanish government) Grant BFU2012-33503, the Consolider Ingenio 2010 program (Grant CSD2007-0015), the Fundación Marcelino Botín (to F. P.), and Grant BFU2011-26722 (to E. d. N.). Recipients of an ICREA Acadèmia (Generalitat de Catalunya). Supported by the MINECO Grant BFU2011-23418. Supported by Agence Nationale de la Recherche (Nucleopol and ODynRib-Jeune chercheur program) and Jeune équipe from Fondation pour la Recherche Médicale.Peer reviewe

Multiple signaling kinases target Mrc1 to prevent genomic instability triggered by transcription-replication conflicts

Conflicts between replication and transcription machineries represent a major source of genomic instability and cells have evolved strategies to prevent such conflicts. However, little is known regarding how cells cope with sudden increases of transcription while replicating. Here, we report the existence of a general mechanism for the protection of genomic integrity upon transcriptional outbursts in S phase that is mediated by Mrc1. The N-terminal phosphorylation of Mrc1 blocked replication and prevented transcription-associated recombination (TAR) and genomic instability during stress-induced gene expression in S phase. An unbiased kinome screening identified several kinases that phosphorylate Mrc1 at the N terminus upon different environmental stresses. Mrc1 function was not restricted to environmental cues but was also required when unscheduled transcription was triggered by low fitness states such as genomic instability or slow growth. Our data indicate that Mrc1 integrates multiple signals, thereby defining a general safeguard mechanism to protect genomic integrity upon transcriptional outbursts.The study was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64437-P and FEDER, BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2014-52333-P and FEDER to E.N.; and BFU2013-42918 and FEDER to A.A.), the Andalusian Government (P12-BIO-1238), ERC2014-ADG669898 TARLOOP, and Worldwide Cancer Research 15-0098 to A.A. and the Catalan Government (2014 SGR 599), and the Fundación Botín, by Banco Santander through its Santander Universities Global Division to F.P. F.P. and E.d.N. are recipients of an ICREA Acadèmia (Generalitat de Catalunya)