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In mammals the melanocortin 4 receptor (Mc4r) signaling system has been mainly associated with the regulation of appetite and energy homeostasis. In fish of the genus Xiphophorus (platyfish and swordtails) puberty onset is genetically determined by a single locus, which encodes the mc4r. Wild populations of Xiphophorus are polymorphic for early and late-maturing individuals. Copy number variation of different mc4r alleles is responsible for the difference in puberty onset. To answer whether this is a special adaptation of the Mc4r signaling system in the lineage of Xiphophorus or a more widely conserved mechanism in teleosts, we studied the role of Mc4r in reproductive biology of medaka (Oryzias latipes), a close relative to Xiphophorus and a well-established model to study gonadal development. To understand the potential role of Mc4r in medaka, we characterized the major features of the Mc4r signaling system (mc4r, mrap2, pomc, agrp1). In medaka, all these genes are expressed before hatching. In adults, they are mainly expressed in the brain. The transcript of the receptor accessory protein mrap2 co-localizes with mc4r in the hypothalamus in adult brains indicating a conserved function of modulating Mc4r signaling. Comparing growth and puberty between wild-type and mc4r knockout medaka revealed that absence of Mc4r does not change puberty timing but significantly delays hatching. Embryonic development of knockout animals is retarded compared to wild-types. In conclusion, the Mc4r system in medaka is involved in regulation of growth rather than puberty.
Opsin 1 and Opsin 2 of the corn smut fungus ustilago maydis are green light-driven proton pumps
(2019)
In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.
Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.
Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.
All physiological processes of ectotherms depend on environmental temperature. Thus, adaptation of physiological mechanisms to the thermal environments is important for achieving optimal performance and fitness. The European Common Frog, Rana temporaria, is widely distributed across different thermal habitats. This makes it an exceptional model for studying the adaptations to different thermal conditions. We raised tadpoles from Germany and Croatia at two constant temperature treatments (15°C, 20°C), and under natural temperature fluctuations (in outdoor treatments), and tested how different developmental temperatures affected developmental traits, that is, length of larval development, morphometrics, and body condition, as well as jumping performance of metamorphs. Our results revealed population‐specific differences in developmental time, body condition, and jumping performance. Croatian frogs developed faster in all treatments, were heavier, in better body condition, and had longer hind limbs and better jumping abilities than German metamorphs. The populations further differed in thermal sensitivity of jumping performance. While metamorphs from Croatia increased their jumping performance with higher temperatures, German metamorphs reached their performance maximum at lower temperatures. These population‐specific differences in common environments indicate local genetic adaptation, with southern populations being better adapted to higher temperatures than those from north of the Alps.
Background
Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.
Methods
Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition.
Results
We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy.
Conclusions
Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.
Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have “gonopodia,” highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation.
Cushing’s disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD’s genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.
It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and – as revealed for perl mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.
Rational drug design of Axl tyrosine kinase type I inhibitors as promising candidates against cancer
(2020)
The high level of Axl tyrosine kinase expression in various cancer cell lines makes it an attractive target for the development of anti-cancer drugs. In this study, we carried out several sets of in silico screening for the ATP-competitive Axl kinase inhibitors based on different molecular docking protocols. The best drug-like candidates were identified, after parental structure modifications, by their highest affinity to the target protein. We found that our newly designed compound R5, a derivative of the R428 patented analog, is the most promising inhibitor of the Axl kinase according to the three molecular docking algorithms applied in the study. The molecular docking results are in agreement with the molecular dynamics simulations using the MM-PBSA/GBSA implicit solvation models, which confirm the high affinity of R5 toward the protein receptor. Additionally, the selectivity test against other kinases also reveals a high affinity of R5 toward ABL1 and Tyro3 kinases, emphasizing its promising potential for the treatment of malignant tumors.
In der vorliegenden Dissertationsarbeit wurden die kardialen Effekte des C-Typ natriuretischen Peptids (CNP) an wildtypischen Mäusen (Studie 1) und an einem neuen genetischen Mausmodell, mit einer Kardiomyozyten-spezifischen Deletion des Guanylyl-Cyclase B (GC-B) Rezeptors (Studie 2) untersucht.
In Studie 1 wurden die Wirkungen von exogenem, synthetischem CNP auf eine durch Druckbelastung-induzierte Herzinsuffizienz in wildtypischen Mäusen (C57Bl6 Hintergrund) untersucht. Dafür wurde CNP parallel zu einer operativen transversen Aortenkonstriktion (TAC) über osmotische Minipumpen in einer Dosierung von 50 ng/kg/min über 14 Tage appliziert. Die 14 Tage TAC führten zu einer ausgeprägten Linksherzhypertrophie. Diese wurde durch exogenes CNP auf zellulärer (verringerte Kardiomyozytenflächen) und molekularer (verringerte BNP mRNA Expression) Ebene signifikant gehemmt. Auch die durch TAC-induzierte linksventrikuläre Dilatation wurde durch exogenes CNP fast vollständig verhindert. Diese kardialen protektiven Effekte von CNP traten ohne eine wesentliche Veränderung des arteriellen Blutdrucks auf. Mögliche mechanistische Ursachen für die schützende Wirkung von CNP könnte die PKG-abhängige Phosphorylierung des sarkomerischen Proteins Titin sein. Eine gesteigerte Phosphorylierung von Titin an der elastischen N2B-Domäne verringert die Steifigkeit der Kardiomyozyten und verbessert somit deren Relaxationsfähigkeit (Hudson 2011). Die erhöhten linksventrikulären Volumina nach TAC (end-diastolische und end-systolische Volumina) wurden möglicherweise durch eine erhöhte Steifigkeit der Kardiomyozyten provoziert. Dies könnte durch den akuten IL-6 mRNA Anstieg nach TAC begünstigt werden, da Kruger et al. einen Zusammenhang zwischen passiver Steifigkeit der Kardiomyozyten und IL-6-Expression postulierten (Kotter 2016, Kruger 2009). Diese Veränderungen wurden durch exogenes CNP verhindert. Es ist wahrscheinlich, dass die CNP-induzierte Phosphorylierung von Titin an Serin 4080 in die Relaxationsfähigkeit der Kardiomyozyten und somit die diastolische Funktion des linken Ventrikels verbesserte.
Aufgrund dieser Beobachtungen wurde in Studie 2 untersucht, ob auch endogenes CNP als parakrines Hormon im Herzen eine TAC-induzierte Herzhypertrophie und die kontraktile Funktion von Kardiomyozyten bei einer hypertensiven Herzerkrankung beeinflussen kann. Dafür wurde ein neues genetisches Mausmodell mit einer Kardiomyozyten-spezifischen Deletion des GC-B Rezeptors generiert (CM GC-B KO). Da vorangegangene Studien in unserer Arbeitsgruppe zeigten, dass die basale CNP-Expression im Herzen sehr gering ist, nach 3-tägiger TAC aber akut ansteigt und nach 14-tägiger TAC wieder abfällt, haben wir CM GC-B KO Mäuse und deren Geschwister-Kontrolltiere an beiden Zeitpunkten nach TAC untersucht. Die TAC führte Genotyp-unabhängig zu einem Anstieg der kardialen Nachlast nach 3 Tagen und weiter nach 14 Tagen. Diese Druckbelastung provozierte eine progressive, signifikante Linksherzhypertrophie.
Allerdings reagierten die CM GC-B KO Mäuse im Vergleich zu den Kontrolltieren bereits nach 3-tägiger TAC mit einer ausgeprägten Kardiomyozyten-Hypertrophie. Zudem beobachteten wir nach 3-tägiger TAC in den Knockout-Mäusen eine Abnahme der Ejektionsfraktion und gleichzeitig eine signifikante Zunahme der beiden linksventrikulären Volumina (end-diastolische und end-systolische Volumen). Diese frühe linksventrikuläre Dilatation wurde in den Kontrolltieren nicht beobachtet. Daraus schlussfolgerten wir, dass endogenes kardiales CNP, dessen Expression zu frühen Zeitpunkten nach Druckbelastung ansteigt, das Herz vor kontraktiler Dysfunktion und Dilatation schützen kann. Um mögliche Mechanismen für die protektive Wirkung von endogenem CNP zu erklären, untersuchten wir die IL-6 mRNA Expression sowie die Titin-Phosphorylierung im Herzen. Der akute Anstieg der IL-6 mRNA Expression nach 3-tägiger TAC in den CM GC-B KO Mäusen korreliert mit der verminderten Phosphorylierung von Titin an der PGK-spezifischen Phosphorylierungsstelle (Serin 4080). Somit könnte der CNP/GC-B/cGMP-Signalweg zu einer Inhibition pro-inflammatorischer Gene beitragen, da der akute IL-6 mRNA Anstieg in den Kontrollen nicht beobachtet wurde. Auch die gesteigerte NOX4 Expression 3 Tage nach TAC, könnte zu der frühen dilatativen Kardiomyopathie in den Knockout-Mäusen beigetragen haben. Die verringerte STAT3 Aktivierung in den CM GC-B KO Mäusen würde laut Literatur zu vermehrter Apoptose führen, indem pro-apoptotische Gene wie Bcl oder Bax vermehrt transkribiert werden. Auch die erhöhte Cxcl-1 mRNA Expression in den Knockout-Mäusen deutet zusammen mit dem IL-6 Anstieg auf vermehrte Entzündungsreaktionen 3 Tage nach TAC hin. Zusammengenommen deuten die Ergebnisse dieser Dissertationsarbeit darauf hin, dass der CNP/GC-B/cGMP-Signalweg in frühen Stadien einer erhöhten kardialen Druckbelastung und der Entstehung einer dilatativen Kardiomyopathie entgegenwirken kann. Die Phosphorylierung des sarkomerischen Proteins Titin und die Hemmung der Expression pro-inflammatorischer Zytokine (speziell IL-6) könnten zu diesem protektiven Effekt beitragen.
Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol’s potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1β) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line.
Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.
To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.
Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network.
Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.
Chlamydia infect millions worldwide and cause infertility and blinding trachoma. Chlamydia trachomatis (C. trachomatis) is an obligate intracellular gram-negative pathogen with a significantly reduced genome. This bacterium shares a unique biphasic lifecycle in which it alternates between the infectious, metabolically inert elementary bodies (EB) and the non-infections, metabolically active replicative reticular bodies (RB).
One of the challenges of working with Chlamydia is its difficult genetic accessibility. In the present work, the high-throughput method TagRNA-seq was used to differentially label transcriptional start sites (TSS) and processing sites (PSS) to gain new insights into the transcriptional landscape of C. trachomatis in a coverage that has never been achieved before. Altogether, 679 TSSs and 1067 PSSs were detected indicating its high transcriptional activity and the need for transcriptional regulation. Furthermore, the analysis of the data revealed potentially new non-coding ribonucleic acids (ncRNA) and a map of transcriptional processing events. Using the upstream sequences, the previously identified σ66 binding motif was detected.
In addition, Grad-seq for C. trachomatis was established to obtain a global interactome of the RNAs and proteins of this intracellular organism. The Grad-Seq data suggest that many of the newly annotated RNAs from the TagRNA-seq approach are present in complexes. Although Chlamydia lack the known RNA-binding proteins (RBPs), e.g. Hfq and ProQ, observations in this work reveal the presence of a previously unknown RBP.
Interestingly, in the gradient analysis it was found that the σ66 factor forms a complex with the RNA polymerase (RNAP). On the other hand, the σ28 factor is unbound. This is in line with results from previous studies showing that most of the genes are under control of σ66. The ncRNA IhtA is known to function via direct base pairing to its target RNA of HctB, and by doing so is influencing the chromatin condensation in Chlamydia. This study confirmed that lhtA is in no complex. On the other hand, the ncRNA ctrR0332 was found to interact with the SNF2 protein ctl0077, a putative helicase. Both molecules co-sedimented in the gradient and were intact after an aptamer-based RNA pull-down. The SWI2/SNF2 class of proteins are nucleosome remodeling complexes. The prokaryotic RapA from E. coli functions as transcription regulator by stimulating the RNAP recycling. This view might imply that the small ncRNA (sRNA) ctrR0332 is part of the global regulation network in C. trachomatis controlling the transition between EBs and RBs via interaction with the SNF2 protein ctl0077.
The present work is the first study describing a global interactome of RNAs and proteins in C. trachomatis providing the basis for future interaction studies in the field of this pathogen.
Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis.
In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbm’s RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbm’s RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts – Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation.
Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingers’ functions.
In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions.
Over the last decade life sciences have made an enormous leap forward. The development of complex analytical instruments, in particular in fluorescence microscopy, has played a decisive role in this. Scientist can now rely on a wide range of imaging techniques that offer different advantages in terms of optical resolution, recording speed or living cell compatibility. With the help of these modern microscopy techniques, multi-protein complexes can be resolved, membrane receptors can be counted, cellular pathways analysed or the internalisation of receptors can be tracked. However, there is currently no universal technique for comprehensive experiment execution that includes dynamic process capture and super resolution imaging on the same target object. In this work, I built a microscope that combines two complementary imaging techniques and enables correlative experiments in living and fixed cells. With an image scanning based laser spot confocal microscope, fast dynamics in several colors with low photodamage of the cells can be recorded. This novel system also has an improved resolution of 170 nm and was thoroughly characterized in this work. The complementary technique is based on single molecule localization microscopy, which can achieve a structural resolution down to 20-30 nm. Furthermore I implemented a microfluidic pump that allows direct interaction with the sample placed on the microscope. Numerous processes such as living cell staining, living cell fixation, immunostaining and buffer exchange can be observed and performed directly on the same cell. Thus, dynamic processes of a cell can be frozen and the structures of interest can be stained and analysed with high-resolution microscopy. Furthermore, I have equipped the detection path of the single molecule technique with an adaptive optical element. With the help of a deformable mirror, imaging functions can be shaped and information on the 3D position of the individual molecules can be extracted.
Der genetische Code beschreibt die Ver- und Entschlüsselung der Erb-information für das universelle Prinzip der Proteinbiosynthese aus einzelnen Aminosäuren. Durch Erweiterung des genetischen Codes lassen sich unna-türliche Aminosäuren (uAA) mit einzigartigen biophysikalischen Eigenschaf-ten ortsspezifisch in Proteine einführen und ermöglichen die spezifische Ma-nipulation von Proteinen.
Die Click-Reaktion zwischen der unnatürlichen Aminosäure TCO*-Lysin und Tetrazin besitzt eine außergewöhnliche Reaktionskinetik (≥800 M-1s-1) und ermöglicht eine spezifische und bioorthogonale Markierung von Bio-
¬molekülen unter physiologischen Bedingungen.
Im Fokus dieser Arbeit stand zunächst die Markierung von Membran-
¬rezeptoren durch Click-Chemie in lebenden Zellen sowie die Untersuchung der Wechselwirkung 22 bekannter und neuartiger Tetrazin-Farbstoff-
Konjugate. Darüber hinaus wurde die Anwendbarkeit von bioorthogonalen Click-Reaktionen für die hochauflösende Fluoreszenzmikroskopie untersucht. Durch Erweiterung des genetischen Codes in Proteine aus der Klasse der ionotropen Glutamatrezeptoren (iGluR), TNF-Rezeptoren oder Mikrotubu-li-assoziierten Proteinen (MAP) wurde ortspezifisch die unnatürliche Amino-säure TCO*-Lysin eingeführt und dadurch die Fluoreszenzmarkierung durch Tetrazin-Farbstoffe ermöglicht. Die direkte chemische Kopplung von TCO an Liganden wie Phalloidin und Docetaxel, welche spezifisch das Aktin-Zytoskelett bzw. Mikrotubuli-Filamente binden können, ermöglichte zudem die Click-Färbungen von fixierten und lebenden Zellen ohne genetische Ver-änderungen der Zielproteine.
Des Weiteren wurden die spektroskopischen Eigenschaften von 22 Tetrazin-Farbstoffen, verteilt über den gesamten sichtbaren Wellenlängenbereich, untersucht. Ein charakteristisches Kennzeichen der Click-Reaktion mit Tet-razin-Farbstoffen ist dabei ihre Fluorogenität. Das Tetrazin fungiert nicht nur als reaktive Gruppe während der Click-Reaktion mit Alkenen, sondern führt in vielen Tetrazin-Farbstoff-Konjugaten zur Fluoreszenzlöschung. Während bei grün-absorbierenden Farbstoffe vor allem FRET-basierte Löschprozesse dominieren, konnte photoinduzierter Elektronentransfer (PET) vom angeregten Farbstoff zum Tetrazin als Hauptlöschmechanismus bei rot-absorbierenden Oxazin- und Rhodamin-Derivaten identifiziert
werden.
Die effiziente und spezifische Markierung aller untersuchten Tetrazin-
Farbstoffe ermöglichte die Visualisierung von Aktin-Filamenten, Mikrotubuli und Membranrezeptoren sowohl durch konventionelle Fluoreszenzmikrosko-pie als auch durch hochauflösende Verfahren, wie z.B. dSTORM, auf Ein-zelmolekülebene. Die unterschiedliche Zellpermeabilität von
Tetrazin-Farbstoffen kann dabei vorteilhaft für die spezifische intra- und extrazelluläre Markierung von Proteinen in fixierten und lebenden Zellen genutzt werden.
The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli.
Background
Xiphophorus fishes are represented by 26 live-bearing species of tropical fish that express many attributes (e.g., viviparity, genetic and phenotypic variation, ecological adaptation, varied sexual developmental mechanisms, ability to produce fertile interspecies hybrids) that have made attractive research models for over 85 years. Use of various interspecies hybrids to investigate the genetics underlying spontaneous and induced tumorigenesis has resulted in the development and maintenance of pedigreed Xiphophorus lines specifically bred for research. The recent availability of the X. maculatus reference genome assembly now provides unprecedented opportunities for novel and exciting comparative research studies among Xiphophorus species.
Results
We present sequencing, assembly and annotation of two new genomes representing Xiphophorus couchianus and Xiphophorus hellerii. The final X. couchianus and X. hellerii assemblies have total sizes of 708 Mb and 734 Mb and correspond to 98 % and 102 % of the X. maculatus Jp 163 A genome size, respectively. The rates of single nucleotide change range from 1 per 52 bp to 1 per 69 bp among the three genomes and the impact of putatively damaging variants are presented. In addition, a survey of transposable elements allowed us to deduce an ancestral TE landscape, uncovered potential active TEs and document a recent burst of TEs during evolution of this genus.
Conclusions
Two new Xiphophorus genomes and their corresponding transcriptomes were efficiently assembled, the former using a novel guided assembly approach. Three assembled genome sequences within this single vertebrate order of new world live-bearing fishes will accelerate our understanding of relationship between environmental adaptation and genome evolution. In addition, these genome resources provide capability to determine allele specific gene regulation among interspecies hybrids produced by crossing any of the three species that are known to produce progeny predisposed to tumor development.
Background
Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.
Results
We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.
Conclusions
This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.
Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated.
Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents (î) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of î and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits.
Das menschliche Gehirn ist ein Organ, das aufgrund seiner Komplexität und zellulären Diversität noch am wenigsten verstanden ist. Eine der Ursachen dafür sind zahlreiche Herausforderungen in diversen neurobiologischen Bild-gebungsverfahren. Erst seit der Erfindung der hochauflösenden Fluoreszenz-mikroskopie ist es möglich, Strukturen unterhalb der Beugungsgrenze zu visua-lisieren und somit eine maximale Auflösung von bis zu 20 nm zu erreichen. Zusätzlich hängt die Fähigkeit, biologische Strukturen aufzulösen, von der Markierungs-größe und -dichte ab. Derzeit ist die häufigste Methode zur Proteinfärbung die indirekte Antikörperfärbung, bei der ein Fluorophor-markierter Sekundärantikörper an einen Epitop-spezifischen Primärantikörper bindet. Dabei kann der Abstand von Zielstruktur und Fluorophor bis zu 30 nm betragen, was eine Auflösungs-verminderung zur Folge haben kann. Aufgrund dessen wurden in dieser Arbeit alternative Markierungsmethoden getestet, um postsynaptische Proteine sicht-bar zu machen.
Zunächst wurde der postsynaptische N-Methyl-D-Aspartat (NMDA)-Rezeptor mit Hilfe konventioneller indirekter Antikörperfärbung markiert. Hier war die NR1-Untereinheit des NMDA-Rezeptors von besonderem Interesse, da diese in der Autoimmunerkrankung Anti-NMDA-Rezeptor-Enzephalitis invol-viert ist.
Patienten dieser seltenen Krankheit bilden Autoantikörper gegen die NR1-Untereinheit, wodurch ein schneller reversibler Verlust der NMDA-Rezeptoren auf der Postsynapse induziert wird. Wichtige Informationen können nicht mehr ausreichend weitergegeben werden, was psychiatrische und neurologi-sche Störungen zur Folge hat. In dieser Arbeit wurden sowohl kommerzielle NR1-Antikörper, als auch rekombinante monoklonale NR1-Antikörper von Patien-ten mit Anti-NMDA-Rezeptor-Enzephalitis getestet. In konfokalen und in hochaufgelösten SIM- (engl. structured illumination microscopy) und dSTORM- (engl. direct stochastic optical reconstruction microscopy) Messun-gen konnten kommerzielle NR1-Antikörper keine erfolgreichen Färbungen erzielen. Dagegen erwiesen sich die rekombinanten monoklonalen NR1-Patientenantikörper als sehr spezifisch, sowohl in primären Neuronen als auch im Hippocampus von murinen Gehirnschnitten und lieferten gute Kolokalisati-onen mit dem postsynaptischen Markerprotein Homer.
Um die optische Auflösung zu verbessern, wurde eine neue Markierungs-methode mit sog. „Super-Binde-Peptiden“ (SBPs) getestet. SBPs sind modifi-zierte Peptide, die erhöhte Affinitäten und Spezifitäten aufweisen und mit ei-ner Größe von ~ 2,5 nm wesentlich kleiner als Antikörper sind. In dieser Arbeit bestätigte sich ein kleines hochspezifisches SPB, das an den Fluoreszenzfarb-stoff Tetra-
methylrhodamin (TMR) gekoppelt ist, als effektiver Marker für das Ankerpro-tein Gephyrin. Gephyrin ist für die Lokalisation und Verankerung einiger post-synaptischer Rezeptoren zuständig, indem es sie mit dem Cytoskelett der Zelle verbindet. SIM-Messungen in primären Neuronen zeigten eine bessere Clus-terrepräsentation bei der Färbung von Gephyrin mit SBPs, als mit Antikörper-färbung. Zusätzlich wurden Kolokalisationsanalysen von Gephyrin zusammen mit dem inhibito-rischen präsynaptischen vesikulären GABA-Transporter VGAT durchgeführt.
Eine weitere Färbemethode stellte die bioorthogonale Click-Färbung durch die Erweiterung des eukaryotischen genetischen Codes (engl. genetic code ex-pansion, GCE) dar. Dabei wurde eine unnatürliche, nicht-kanonische Amino-säure (engl. non-canonical amino acid, ncAA) ins Zielprotein eingebaut und in Kombination mit der Click-Chemie ortsspezifisch mit organischen Tetrazin-Farbstoff-Konjugaten angefärbt. Organische Fluorophore haben den Vorteil, dass sie mit einer Größe von 0,5 – 2 nm sehr klein sind und damit die natürli-chen Funktionen der Proteine in der Zelle kaum beeinflussen. In dieser Arbeit wurde zum ersten Mal gezeigt, dass der tetramere postsynaptische NMDA-Rezeptor durch die Amber-Supres-sionsmethode bioorthogonal angefärbt werden konnte. Aus sieben verschiede-nen Amber-Mutanten der NR1-Untereinheit stellte sich die Y392TAG-NR1-Mutante als diejenige mit der besten Proteinexpression, Färbeeffizienz und rezeptorfunktionalität heraus. Dies konnte durch Fluoreszenzmikroskopie- und Whole-Cell Patch-Clamp-Experimenten gezeigt werden. Die bioorthogo-nale Click-Färbung durch GCE eignete sich für die Färbung des NMDA-Rezeptors in verschiedenen Zelllinien, mit unterschiedlichen Tetrazin-Farbstoff-Konjugaten und für Lebendzellexperimente. In dSTORM-Messungen erwies sich das Tetrazin-Cy5-Farbstoff-Konjugat als ideal aufgrund seiner Grö-ße, Photostabilität, Helligkeit und seines geeigneten Blinkverhaltens, sodass eine homogene NMDA-Rezeptorverteilung auf der Zellmembran gezeigt wer-den konnte. NR1-Antikörperfärbungen wiesen dagegen starke Clusterbildun-gen auf. Die Ergebnisse konnten belegen, dass kleinere Farbstoffe eine deut-lich bessere Zugänglichkeit zu ihrem Zielprotein haben und somit besser für die hochauflösende Fluoreszenzmikroskopie geeignet sind.
Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.
Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies.
This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania.
Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used.
Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients.
Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today.
Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity.
In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.
Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\
Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information $per$ $definitionem$.\\
Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\
The work presented in this thesis is part of the ongoing effort to translate those $per$ $se$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\
During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\
In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\
This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\
The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\
In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\
Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\
Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\
With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a $z$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\
Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\
Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research.
Social immunity is a key factor for honeybee health, including behavioral defense strategies such as the collective use of antimicrobial plant resins (propolis). While laboratory data repeatedly show significant propolis effects, field data are scarce, especially at the colony level. Here, we investigated whether propolis, as naturally deposited in the nests, can protect honeybees against ectoparasitic mites Varroa destructor and associated viruses, which are currently considered the most serious biological threat to European honeybee subspecies, Apis mellifera, globally. Propolis intake of 10 field colonies was manipulated by either reducing or adding freshly collected propolis. Mite infestations, titers of deformed wing virus (DWV) and sacbrood virus (SBV), resin intake, as well as colony strength were recorded monthly from July to September 2013. We additionally examined the effect of raw propolis volatiles on mite survival in laboratory assays. Our results showed no significant effects of adding or removing propolis on mite survival and infestation levels. However, in relation to V. destructor, DWV titers increased significantly less in colonies with added propolis than in propolis-removed colonies, whereas SBV titers were similar. Colonies with added propolis were also significantly stronger than propolis-removed colonies. These findings indicate that propolis may interfere with the dynamics of V. destructor-transmitted viruses, thereby further emphasizing the importance of propolis for honeybee health.
Numerous studies revealed a positive relationship between biodiversity and ecosystem functioning, suggesting that biodiverse environments may not only enhance ecosystem processes, but also benefit individual ecosystem members by, for example, providing a higher diversity of resources. Whether and how the number of available resources affects resource collection and subsequently consumers (e.g., through impacting functions associated with resources) have, however, been little investigated, although a better understanding of this relationship may help explain why the abundance and richness of many animal species typically decline with decreasing plant (resource) diversity. Using a social bee species as model (Tetragonula carbonaria), we investigated how plant species richness—recorded for study sites located in different habitats—and associated resource abundance affected the diversity and functionality (here defined as nutritional content and antimicrobial activity) of resources (i.e., pollen, nectar, and resin) collected by a generalist herbivorous consumer. The diversity of both pollen and resin collected strongly increased with increasing plant/tree species richness, while resource abundance was only positively correlated with resin diversity. These findings suggest that bees maximize resource diversity intake in (resource) diverse habitats. Collecting more diverse resources did, however, not increase their functionality, which appeared to be primarily driven by the surrounding (plant) source community in our study. In generalist herbivores, maximizing resource diversity intake may therefore primarily secure collection of sufficient amounts of resources across the entire foraging season, but it also ensures that the allocated resources meet all functional needs. Decreasing available resource diversity may thus impact consumers primarily by reduced resource abundance, but also by reduced resource functionality, particularly when resources of high functionality (e.g., from specific plant species) become scarce.
Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC\(_{50}\)< 3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.
Theory predicts that males and females should often join the mating pool at different times (sexual dimorphism in timing of emergence [SDT]) as the degree of SDT affects female mating success. We utilize an analytical model to explore (1) how important SDT is for female mating success, (2) how mating success might change if either sex's mortality (abruptly) increases, and (3) to what degree evolutionary responses in SDT may be able to mitigate the consequences of such mortality increase. Increasing male pre‐mating mortality has a non‐linear effect on the fraction of females mated: The effect is initially weak, but at some critical level a further increase in male mortality has a stronger effect than a similar increase in female mortality. Such a change is expected to impose selection for reduced SDT. Increasing mortality during the mating season has always a stronger effect on female mating success if the mortality affects the sex that emerges first. This bias results from the fact that enhancing mortality of the earlier emerging sex reduces female–male encounter rates. However, an evolutionary response in SDT may effectively mitigate such consequences. Further, if considered independently for females and males, the predicted evolutionary response in SDT could be quite dissimilar. The difference between female and male evolutionary response in SDT leads to marked differences in the fraction of fertilized females under certain conditions. Our model may provide general guidelines for improving harvesting of populations, conservation management of rare species under altered environmental conditions, or maintaining long‐term efficiency of pest‐control measures.
Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.
The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.
Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.
The instructive component of waggle dance communication has been shown to increase resource uptake of Apis mellifera colonies in highly heterogeneous resource environments, but an assessment of its relevance in temperate landscapes with different levels of resource heterogeneity is currently lacking. We hypothesized that the advertisement of resource locations via dance communication would be most relevant in highly heterogeneous landscapes with large spatial variation of floral resources. To test our hypothesis, we placed 24 Apis mellifera colonies with either disrupted or unimpaired instructive component of dance communication in eight Central European agricultural landscapes that differed in heterogeneity and resource availability. We monitored colony weight change and pollen harvest as measure of foraging success. Dance disruption did not significantly alter colony weight change, but decreased pollen harvest compared to the communicating colonies by 40%. There was no general effect of resource availability on nectar or pollen foraging success, but the effect of landscape heterogeneity on nectar uptake was stronger when resource availability was high. In contrast to our hypothesis, the effects of disrupted bee communication on nectar and pollen foraging success were not stronger in landscapes with heterogeneous compared to homogenous resource environments. Our results indicate that in temperate regions intra-colonial communication of resource locations benefits pollen foraging more than nectar foraging, irrespective of landscape heterogeneity. We conclude that the so far largely unexplored role of dance communication in pollen foraging requires further consideration as pollen is a crucial resource for colony development and health.
Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.
Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems.
We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.
High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization.
Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskulären Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverzügliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gewährleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu ermöglichen und eine automatisierte Lösung für ähnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros für ImageJ, eines für die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte ermöglicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskulärer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalität der Software bewiesen werden. Die Analyse der neuromuskulären Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen.
Im zweiten Teil der Arbeit wurden neuromuskulärer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans über mehrere Monate ohne Nahrungsaufnahme überleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core“-Vesikel (CCV) und der „dense-core“-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning“-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erhöhte Anzahl von „dense-core“-Vesikeln, sowie eine veränderte Lokalisation der DCV in Dauerlarven festgestellt werden.
Im dritten Teil dieser Arbeit wurde untersucht ob die für synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierfür eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss.
The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.
Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
A recent study by Peng and Yang in Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22–180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains.
The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.
Cytotoxic T lymphocytes are effector CD8\(^{+}\) T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1\(^{-/-}\) cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1\(^{-/-}\) CD8\(^{+}\) T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1\(^{-/-}\), but not Nfatc2\(^{-/-}\) CD8\(^{+}\) T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.