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Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability.
SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype.
The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1.
Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage.
Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis.
To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.
BMPs vermitteln ihre zellulären Effekte durch Rekrutierung und Aktivierung von zwei Typen spezifischer, membranständiger Rezeptoren. Die genauen Mechanismen der Rezeptorakivierung und die Komposition eines funktionellen, signalvermittelnden Komplexes auf der Zelloberfläche sind in den letzten Jahren genau untersucht worden. Die dimere Natur aller BMPs, die Promiskuitivität der BMPs sowie der entsprechenden Rezeptoren und die unterschiedlichen Rezeptorkonformationen (PFC, BISC) erschweren jedoch die experimentelle Zugänglichkeit dieser Proteinfamilie. Um den Einfluss der Membranverankerung der Rezeptoren auf deren Affinität zu einzelnen Liganden zu untersuchen, wurden verschiedene Methoden evaluiert, die eine quantitative Kopplung an Plasmamembranen ermöglichten. Die BMP Rezeptorektodomänen wurden u.a. mittels einer lysin-spezifischen Kopplung lipidiert, oder aber als His6-Ektodomänen an membranintegrierte Chelatlipide gekoppelt.
Recently reported insect declines have raised both political and social concern. Although the declines have been attributed to land use and climate change, supporting evidence suffers from low taxonomic resolution, short time series, a focus on local scales, and the collinearity of the identified drivers. In this study, we conducted a systematic assessment of insect populations in southern Germany, which showed that differences in insect biomass and richness are highly context dependent. We found the largest difference in biomass between semi-natural and urban environments (-42%), whereas differences in total richness (-29%) and the richness of threatened species (-56%) were largest from semi-natural to agricultural environments. These results point to urbanization and agriculture as major drivers of decline. We also found that richness and biomass increase monotonously with increasing temperature, independent of habitat. The contrasting patterns of insect biomass and richness question the use of these indicators as mutual surrogates. Our study provides support for the implementation of more comprehensive measures aimed at habitat restoration in order to halt insect declines.
Recent reports on insect decline have highlighted the need for long‐term data on insect communities towards identifying their trends and drivers.
With the launch of many new insect monitoring schemes to investigate insect communities over large spatial and temporal scales, Malaise traps have become one of the most important tools due to the broad spectrum of species collected and reduced capture bias through passive sampling of insects day and night. However, Malaise traps can vary in size, shape, and colour, and it is unknown how these differences affect biomass, species richness, and composition of trap catch, making it difficult to compare results between studies.
We compared five Malaise trap types (three variations of the Townes and two variations of the Bartak Malaise trap) to determine their effects on biomass and species richness as identified by metabarcoding.
Insect biomass varied by 20%–55%, not strictly following trap size but varying with trap type. Total species richness was 20%–38% higher in the three Townes trap models compared to the Bartak traps. Bartak traps captured lower richness of highly mobile taxa but increased richness of ground‐dwelling taxa. The white roofed Townes trap captured a higher richness of pollinators.
We find that biomass, total richness, and taxa group specific richness are all sensitive to Malaise trap type. Trap type should be carefully considered and aligned to match monitoring and research questions. Additionally, our estimates of trap type effects can be used to adjust results to facilitate comparisons across studies.
ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1
Mit fortschreitender chronischer Niereninsuffizienz kommt es zur Akkumulation von Urämietoxinen und im Endstadium unbehandelt zum Tod im sogenannten Urämischen Syndrom. Die Blutreinigung erfolgt bei der am häufigsten verwendeten Form der Nierenersatztherapie, der Hämodialyse, nur unzureichend. Die Folge ist eine erhöhte Morbidität und Mortalität der betroffenen Patienten. Bei der Hämodialyse werden nur Urämietoxine bis zu einer Größe von 20 kDa über die im Dialysator eingesetzten Hohlfaserdialysemembranen diffusiv und konvektiv semiselektiv nach Größenausschluss entfernt. Proteingebundene Urämietoxine, deren effektive Größe durch die Bindung an Transportproteine wie beispielsweise Albumin die Trennschärfe der Dialysemembranen übersteigt, werden retiniert. In-vivo werden proteingebundene Urämietoxine im proximalen Tubulus, einem Teil des tubulären Systems des Nephrons, sekretorisch eliminiert.
Im Rahmen der vorliegenden Promotionsarbeit wurden die ersten Entwicklungsschritte auf dem Weg zu einem sogenannten bio-artifiziellen Tubulus evaluiert. Der angedachte biohybride Filter sollte aus einer Ko-Kultur funktionaler humaner proximaler Tubuluszellen und humaner Endothelzellen (HUVEC) auf synthetischen Hohlfasermembranen bestehen und könnte während der Hämodialyse als zusätzlicher Reinigungsschritt angewendet werden, um unter anderem proteingebundene Urämietoxine effektiv durch aktiven Transport aus dem Blut der Patienten zu entfernen. Die Differenzierung der proximalen Tubuluszellen erfolgte dabei aus adulten adipozytären mesenchymalen Stammzellen (ASC), deren Herkunft eine spätere autologe Behandlung ermöglicht. Die Ko-Kultur mit Endothelzellen wurde zur potentiellen Steigerung der Sekretion proteingebundener Urämietoxine verwendet.
In der vorliegenden Arbeit konnten ASCs durch eine Kombination der löslichen Differenzierungsfaktoren All-Trans-Retinoinsäure (ATRA), Aktivin A und BMP-7 erfolgreich in Zytokeratin 18-exprimierende Zellen differenziert werden, wodurch die erwünschte epitheliale Differenzierung bestätigt wurde. Die Expression funktionaler Proteine, wie das für den Wassertransport relevante Aquaporin 1 oder auch der Na+-/K+-ATPase, konnte in dieser Arbeit bereits vor der Differenzierung nachgewiesen werden. Im nächsten Schritt wurde erfolgreich gezeigt, dass eine simultane, qualitativ hochwertige Ko-Kultur von ASCs und HUVECs auf der mit dem extrazellulären Matrixprotein Fibronektin modifizierten Innen- bzw. Außenseite von synthetischen Hohlfasermembranen aus Polypropylen bzw. Polyethersulfon möglich ist. Die Viabilität beider Zelltypen wurde dabei durch die Verwendung eines für die Ko-Kultur entwickelten Nährmediums erreicht, in welchem die Proliferation von ASCs bei gleichzeitiger Aufrechterhaltung ihrer Stammzelleigenschaften deutlich erhöht war.
Die in dieser Arbeit erzielten Ergebnisse stellen eine aussichtsreiche Basis für einen bio-artifiziellen renalen Tubulus dar. Weitere Entwicklungsschritte, wie die Differenzierung der ASCs zu proximalen Tubuluszellen im 3D-Bioreaktor einschließlich ihrer funktionalen Charakterisierung anhand Tubulusepithel-spezifischer Transporter, sind erforderlich, be-vor erste funktionale Experimente vor dem „Upscaling“ auf klinisch verwendbare Module möglich sind.
Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections
(2015)
Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, \(\Delta\)sigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.
Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1
(2012)
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.