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Sonstige beteiligte Institutionen
- DNA Analytics Core Facility, Biocenter, University of Wuerzburg, Wuerzburg, Germany (1)
- EMBL, Structural and Computational Biology Unit, Heidelberg, Germany (1)
- IZKF Laboratory for Microarray Applications, University Hospital of Wuerzburg, Wuerzburg, Germany (1)
- Microarray Core Unit, Interdisciplinary Center for Clinical Science, University of Würzburg, Versbacher Straße, Würzburg 97080, Germany (1)
Background
Angiogenesis represents a highly multi-factorial and multi-cellular complex (patho-) physiologic event involving endothelial cells, tumor cells in malignant conditions, as well as bone marrow derived cells and stromal cells. One main driver is vascular endothelial growth factor (VEGFA), which is known to interact with endothelial cells as a survival and mitogenic signal. The role of VEGFA on tumor cells and /or tumor stromal cell interaction is less clear. Condition specific (e.g. hypoxia) or tumor specific expression of VEGFA, VEGF receptors and co-receptors on tumor cells has been reported, in addition to the expression on the endothelium. This suggests a potential paracrine/autocrine loop that could affect changes specific to tumor cells.
Methods
We used the monoclonal antibody against VEGFA, bevacizumab, in various in vitro experiments using cell lines derived from different tumor entities (non small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC) and renal cell carcinoma (RCC)) in order to determine if potential VEGFA signaling could be blocked in tumor cells. The experiments were done under hypoxia, a major inducer of VEGFA and angiogenesis, in an attempt to mimic the physiological tumor condition. Known VEGFA induced endothelial biological responses such as proliferation, migration, survival and gene expression changes were evaluated.
Results
Our study was able to demonstrate expression of VEGF receptors on tumor cells as well as hypoxia regulated angiogenic gene expression. In addition, there was a cell line specific effect in tumor cells by VEGFA blockade with bevacizumab in terms of proliferation; however overall, there was a limited measurable consequence of bevacizumab therapy detected by migration and survival.
Conclusion
The present study showed in a variety of in vitro experiments with several tumor cell lines from different tumor origins, that by blocking VEGFA with bevacizumab, there was a limited autocrine or cell-autonomous function of VEGFA signaling in tumor cells, when evaluating VEGFA induced downstream outputs known in endothelial cells.
PTEN induced kinase 1 (PINK1) is a serine/threonine kinase in the outer membrane of mitochondria (OMM), and known as a responsible gene of Parkinson's disease (PD). The precursor of PINK1 is synthesized in the cytosol and then imported into the mitochondria via the translocase of the OMM (TOM) complex. However, a large part of PINK1 import mechanism remains unclear. In this study, we examined using cell-free system the mechanism by which PINK1 is targeted to and assembled into mitochondria. Surprisingly, the main component of the import channel, Tom40 was not necessary for PINK1 import. Furthermore, we revealed that the import receptor Tom70 is essential for PINK1 import. In addition, we observed that although PINK1 has predicted mitochondrial targeting signal, it was not processed by the mitochondrial processing peptidase. Thus, our results suggest that PINK1 is imported into mitochondria by a unique pathway that is independent of the TOM core complex but crucially depends on the import receptor Tom70.
In the first decade of the 20th century, a horse named Hans drew worldwide attention in Berlin as the first and most famous “speaking” and thinking animal. Hans solved calculations by tapping numbers or letters with his hoof in order to answer questions. Later on, it turned out that the horse was able to give the correct answer by reading the microscopic signals in the face of the questioning person. This observation caused a revolution and as a consequence, experimenters avoided strictly any face-to-face contact in studies about cognitive abilities of animals—a fundamental lesson that is still not applied rigorously.
The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level.
Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.
Background
The actin cytoskeleton is essential for many physiological processes of eukaryotic cells. The emergence of new actin fibers is initiated by actin nucleators. Whereas most of them are evolutionary old, the cordon-bleu actin nucleator is classified as vertebrate specific.
Findings
Using sensitive methods for sequence similarity detection, we identified homologs of cordon-bleu not only in non-vertebrate chordates but also in arthropods, molluscs, annelids and platyhelminthes. These genes contain only a single WH2 domain and therefore resemble more the vertebrate cordon-bleu related 1 protein than the three WH2 domain containing cordon-bleu. Furthermore, we identified a homolog of the N-terminal, ubiquitin like, cobl domain of cordon-bleu in the cnidarian Nematostella vectensis.
Conclusion
Our results suggest that the ur-form of the cordon-bleu protein family evolved already with the emergence of the bilateria by the combination of existing cobl and WH2 domains. Following a vertebrate specific gene-duplication, one copy gained two additional WH2 domains leading to the actin nucleating cordon-bleu. The function of the ur-form of the cordon-bleu protein family is so far unknown. The identification of a homolog in the model organism Drosophila melanogaster could facilitate its experimental characterization.
The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env\((\Delta CT)\) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.
Host colonization by lymphotropic \(\gamma\)-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of \(\gamma\)-cherpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic \(\gamma\)-herpesvirus MuHV- 4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B- cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological posttranslational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly- ubiquitination mediated via the viral E3 ubiquitin- ligase mLANA protein. \(EC_5S^{mLANA}\) modulates cellular control of Myc turnover by antagonizing \(SCF^{Fbw7}\) mediated proteasomal degradation of Myc, mimicking \(SCF^{\beta-TrCP}\). The findings here reported reveal that modulation of Myc is essential for \(\gamma\)-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.
Background
The knowledge of metabolic pathways and fluxes is important to understand the adaptation of organisms to their biotic and abiotic environment. The specific distribution of stable isotope labelled precursors into metabolic products can be taken as fingerprints of the metabolic events and dynamics through the metabolic networks. An open-source software is required that easily and rapidly calculates from mass spectra of labelled metabolites, derivatives and their fragments global isotope excess and isotopomer distribution.
Results
The open-source software “Least Square Mass Isotopomer Analyzer” (LS-MIDA) is presented that processes experimental mass spectrometry (MS) data on the basis of metabolite information such as the number of atoms in the compound, mass to charge ratio (m/e or m/z) values of the compounds and fragments under study, and the experimental relative MS intensities reflecting the enrichments of isotopomers in 13C- or 15 N-labelled compounds, in comparison to the natural abundances in the unlabelled molecules. The software uses Brauman’s least square method of linear regression. As a result, global isotope enrichments of the metabolite or fragment under study and the molar abundances of each isotopomer are obtained and displayed.
Conclusions
The new software provides an open-source platform that easily and rapidly converts experimental MS patterns of labelled metabolites into isotopomer enrichments that are the basis for subsequent observation-driven analysis of pathways and fluxes, as well as for model-driven metabolic flux calculations.
Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane.
Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding.
Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.
This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
Ranging from dwarfs to giants, the species of honeybees show remarkable differences in body size that have placed evolutionary constrains on the size of sensory organs and the brain. Colonies comprise three adult phenotypes, drones and two female castes, the reproductive queen and sterile workers. The phenotypes differ with respect to tasks and thus selection pressures which additionally constrain the shape of sensory systems. In a first step to explore the variability and interaction between species size-limitations and sex and caste-specific selection pressures in sensory and neural structures in honeybees, we compared eye size, ommatidia number and distribution of facet lens diameters in drones, queens and workers of five species (Apis andreniformis, A. florea, A. dorsata, A. mellifera, A. cerana). In these species, male and female eyes show a consistent sex-specific organization with respect to eye size and regional specialization of facet diameters. Drones possess distinctly enlarged eyes with large dorsal facets. Aside from these general patterns, we found signs of unique adaptations in eyes of A. florea and A. dorsata drones. In both species, drone eyes are disproportionately enlarged. In A. dorsata the increased eye size results from enlarged facets, a likely adaptation to crepuscular mating flights. In contrast, the relative enlargement of A. florea drone eyes results from an increase in ommatidia number, suggesting strong selection for high spatial resolution. Comparison of eye morphology and published mating flight times indicates a correlation between overall light sensitivity and species-specific mating flight times. The correlation suggests an important role of ambient light intensities in the regulation of species-specific mating flight times and the evolution of the visual system. Our study further deepens insights into visual adaptations within the genus Apis and opens up future perspectives for research to better understand the timing mechanisms and sensory physiology of mating related signals.
The Chaco leaf-cutting ant Atta vollenweideri (Forel) inhabits large and deep subterranean nests composed of a large number of fungus and refuse chambers. The ants dispose of the excavated soil by forming small pellets that are carried to the surface. For ants in general, the organisation of underground soil transport during nest building remains completely unknown. In the laboratory, we investigated how soil pellets are formed and transported, and whether their occurrence influences the spatial organisation of collective digging. Similar to leaf transport, we discovered size matching between soil pellet mass and carrier mass. Workers observed while digging excavated pellets at a rate of 26 per hour. Each excavator deposited its pellets in an individual cluster, independently of the preferred deposition sites of other excavators. Soil pellets were transported sequentially over 2 m, and the transport involved up to 12 workers belonging to three functionally distinct groups: excavators, several short-distance carriers that dropped the collected pellets after a few centimetres, and long-distance, last carriers that reached the final deposition site. When initiating a new excavation, the proportion of long-distance carriers increased from 18% to 45% within the first five hours, and remained unchanged over more than 20 hours. Accumulated, freshly-excavated pellets significantly influenced the workers' decision where to start digging in a choice experiment. Thus, pellets temporarily accumulated as a result of their sequential transport provide cues that spatially organise collective nest excavation.
Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria
(2013)
β-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial β-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial β-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial β-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal β-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the β-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial β-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of β-barrel proteins plays a role in directing these proteins to mitochondria.
More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.
Author Summary:
Human herpesviruses (HHVs) can reside in a lifelong non-infectious state displaying limited activity in their host and protected from immune responses. One possible way by which HHV-6 achieves this state is by integrating into the telomeric ends of human chromosomes, which are highly repetitive sequences that protect the ends of chromosomes from damage. Various stress conditions can reactivate latent HHV-6 thus increasing the severity of multiple human disorders. Recently, we have identified Chlamydia infection as a natural cause of latent HHV-6 reactivation. Here, we have sought to elucidate the molecular mechanism of HHV-6 reactivation. HHV-6 efficiently utilizes the well-organized telomere maintenance machinery of the host cell to exit from its inactive state and initiate replication to form new viral DNA. We provide experimental evidence that the shortening of telomeres, as a consequence of interference with telomere maintenance, triggers the release of the integrated virus from the chromosome. Our data provide a mechanistic basis to understand HHV-6 reactivation scenarios, which in light of the high prevalence of HHV-6 infection and the possibility of chromosomal integration of other common viruses like HHV-7 have important medical consequences for several million people worldwide.
Background
Phytoplankton communities are often used as a marker for the determination of fresh water quality. The routine analysis, however, is very time consuming and expensive as it is carried out manually by trained personnel. The goal of this work is to develop a system for an automated analysis.
Results
A novel open source system for the automated recognition of phytoplankton by the use of microscopy and image analysis was developed. It integrates the segmentation of the organisms from the background, the calculation of a large range of features, and a neural network for the classification of imaged organisms into different groups of plankton taxa. The analysis of samples containing 10 different taxa showed an average recognition rate of 94.7% and an average error rate of 5.5%. The presented system has a flexible framework which easily allows expanding it to include additional taxa in the future.
Conclusions
The implemented automated microscopy and the new open source image analysis system - PlanktoVision - showed classification results that were comparable or better than existing systems and the exclusion of non-plankton particles could be greatly improved. The software package is published as free software and is available to anyone to help make the analysis of water quality more reproducible and cost effective.
Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.
Plant communities in the European Alps are assumed to be highly affected by climate change, as the temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010, an experiment simulating advanced and delayed snowmelt as well as a drought event was conducted along an altitudinal transect approximately every 250 m (600–2000 m above sea level) in the Berchtesgaden National Park, Germany. The study showed that flower phenology was strongly affected by altitude; however, there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes may be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes, as changes may be more pronounced at higher altitudes. These data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
Animals face highly complex and dynamic olfactory stimuli in their natural environments, which require fast and reliable olfactory processing. Parallel processing is a common principle of sensory systems supporting this task, for example in visual and auditory systems, but its role in olfaction remained unclear. Studies in the honeybee focused on a dual olfactory pathway. Two sets of projection neurons connect glomeruli in two antennal-lobe hemilobes via lateral and medial tracts in opposite sequence with the mushroom bodies and lateral horn. Comparative studies suggest that this dual-tract circuit represents a unique adaptation in Hymenoptera. Imaging studies indicate that glomeruli in both hemilobes receive redundant sensory input. Recent simultaneous multi-unit recordings from projection neurons of both tracts revealed widely overlapping response profiles strongly indicating parallel olfactory processing. Whereas lateral-tract neurons respond fast with broad (generalistic) profiles, medial-tract neurons are odorant specific and respond slower. In analogy to “what-” and “where” subsystems in visual pathways, this suggests two parallel olfactory subsystems providing “what-” (quality) and “when” (temporal) information. Temporal response properties may support across-tract coincidence coding in higher centers. Parallel olfactory processing likely enhances perception of complex odorant mixtures to decode the diverse and dynamic olfactory world of a social insect.
Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).
Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.
Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.