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Institute
- Institut für Biochemie und Biologie (94) (remove)
How predictable is the next move of an animal? Specifically, which factors govern the short- and long-term motion patterns and the overall dynamics of land-bound, plant-eating animals in general and ruminants in particular? To answer this question, we here study the movement dynamics of springbok antelopes Antidorcas marsupialis. We propose several complementary statistical-analysis techniques combined with machine-learning approaches to analyze—across multiple time scales—the springbok motion recorded in long-term GPS tracking of collared springboks at a private wildlife reserve in Namibia. As a result, we are able to predict the springbok movement within the next hour with a certainty of about 20%. The remaining about 80% are stochastic in nature and are induced by unaccounted factors in the modeling algorithm and by individual behavioral features of springboks. We find that directedness of motion contributes approximately 17% to this predicted fraction. We find that the measure for directedeness is strongly dependent on the daily cycle of springbok activity. The previously known daily affinity of springboks to their water points, as predicted from our machine-learning algorithm, overall accounts for only about 3% of this predicted deterministic component of springbok motion. Moreover, the resting points are found to affect the motion of springboks at least as much as the formally studied effects of water points. The generality of these statements for the motion patterns and their underlying behavioral reasons for other ruminants can be examined on the basis of our statistical-analysis tools in the future.
Step selection analysis (SSA) is a common framework for understanding animal movement and resource selection using telemetry data. Such data are, however, inherently autocorrelated in space, a complication that could impact SSA‐based inference if left unaddressed. Accounting for spatial correlation is standard statistical practice when analysing spatial data, and its importance is increasingly recognized in ecological models (e.g. species distribution models). Nonetheless, no framework yet exists to account for such correlation when analysing animal movement using SSA.
Here, we extend the popular method integrated step selection analysis (iSSA) by including a Gaussian field (GF) in the linear predictor to account for spatial correlation. For this, we use the Bayesian framework R‐INLA and the stochastic partial differential equations (SPDE) technique.
We show through a simulation study that our method provides accurate fixed effects estimates, quantifies their uncertainty well and improves the predictions. In addition, we demonstrate the practical utility of our method by applying it to three wolverine (Gulo gulo) tracks.
Our method solves the problems of assuming spatially independent residuals in the SSA framework. In addition, it offers new possibilities for making long‐term predictions of habitat usage.
The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid–lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.
Animal societies are structured of dominance hierarchy (DH). DH can be viewed as networks and analyzed by graph theory. We study the impact of state-dependent feedback (winner-loser effect) on the emergence of local dominance structures after pairwise contests between initially equal-ranking members (equal resource-holding-power, RHP) of small and large social groups. We simulated pairwise agonistic contests between individuals with and without a priori higher RHP by Monte-Carlo-method. Random pairwise contests between equal-ranking competitors result in random dominance structures (‘Null variant’) that are low in transitive triads and high in pass along triads; whereas state-dependent feedback (‘Winner-loser variant’) yields centralized ‘star’ structured DH that evolve from competitors with initially equal RHP and correspond to hierarchies that evolve from keystone individuals. Monte-Carlo simulated DH following state-dependent feedback show motif patterns very similar to those of a variety of natural DH, suggesting that state-dependent feedback plays a pivotal role in robust self-organizing phenomena that transcend the specifics of the individual. Self-organization based on state-dependent feedback leads to social structures that correspond to those resulting from pre-existing keystone individuals. As the efficiency of centralized social networks benefits both, the individual and the group, centralization of social networks appears to be an important evolutionary goal.
Small temporary wetlands, like kettle holes, provide many valuable ecosystem functions and serve as refuge habitats in otherwise monotonous agricultural landscapes. However, the mechanisms that maintain biodiversity in these habitats are still poorly understood. In this study, we investigate how three taxa (vascular plants, ground beetles and spiders) respond to small-scale flooding and disturbance gradients in kettle holes as well as kettle hole area. For this purpose, we determined total, hygrophilic and red list species richness for all taxa and activity density for arthropods along transects extending from the edge towards the center of kettle holes. Furthermore, we calculated the community-weighted mean body size for arthropods and seed mass for plants as surrogates for the ability to respond to disturbance. Our analyses revealed that in particular plants and ground beetles showed strong responses along the small-scale spatial gradient. Total plant species richness decreased towards the center, while hygrophilic plant species increased. In contrast, both total and hygrophilic species richness of ground beetles increased towards the center. Spiders showed similar responses as ground beetles, but less pronounced. We found no evidence that disturbance at the edge of kettle holes leads to smaller body sizes or seed masses. However, arthropods in adjacent arable fields (one meter from the kettle hole edge) were particularly small. Kettle hole area had only weak effects on plants, but not on arthropods. Our study indicates that differences in the depth at the drier edge and the moist, regularly flooded center have a large and taxon-dependent influence on the species composition. Therefore, small-scale heterogeneity seems to be an important predictor for the maintenance of species diversity.
Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO2-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO2 capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD+ vs. NADP+ reduction. Starting from the NAD+-specific wild-type RcFDH, key residues were exchanged to enable NADP+ binding on the basis of the NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys157) in the β-subunit of the enzyme is essential for the binding of NAD+. RcFDH variants that had Glu259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP+. The FdsBL279R and FdsBK276A variants also showed activity with NADP+. Kinetic parameters for all the variants were determined and tested for activity in CO2 reduction. The variants were able to reduce CO2 using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.
The deficiency of a (bio)chemical reaction network can be conceptually interpreted as a measure of its ability to support exotic dynamical behavior and/or multistationarity. The classical definition of deficiency relates to the capacity of a network to permit variations of the complex formation rate vector at steady state, irrespective of the network kinetics. However, the deficiency is by definition completely insensitive to the fine details of the directionality of reactions as well as bounds on reaction fluxes. While the classical definition of deficiency can be readily applied in the analysis of unconstrained, weakly reversible networks, it only provides an upper bound in the cases where relevant constraints on reaction fluxes are imposed. Here we propose the concept of effective deficiency, which provides a more accurate assessment of the network’s capacity to permit steady state variations at the complex level for constrained networks of any reversibility patterns. The effective deficiency relies on the concept of nonstoichiometric balanced complexes, which we have already shown to be present in real-world biochemical networks operating under flux constraints. Our results demonstrate that the effective deficiency of real-world biochemical networks is smaller than the classical deficiency, indicating the effects of reaction directionality and flux bounds on the variation of the complex formation rate vector at steady state.
Introduction Flux phenotypes from different organisms and growth conditions allow better understanding of differential metabolic networks functions. Fluxes of metabolic reactions represent the integrated outcome of transcription, translation, and post-translational modifications, and directly affect growth and fitness. However, fluxes of intracellular metabolic reactions cannot be directly measured, but are estimated via metabolic flux analysis (MFA) that integrates data on isotope labeling patterns of metabolites with metabolic models. While the application of metabolomics technologies in photosynthetic organisms have resulted in unprecedented data from 13CO2-labeling experiments, the bottleneck in flux estimation remains the application of isotopically nonstationary MFA (INST-MFA). INST-MFA entails fitting a (large) system of coupled ordinary differential equations, with metabolite pools and reaction fluxes as parameters. Here, we focus on the Calvin-Benson cycle (CBC) as a key pathway for carbon fixation in photosynthesizing organisms and ask if approaches other than classical INST-MFA can provide reliable estimation of fluxes for reactions comprising this pathway.
Methods First, we show that flux estimation with the labeling patterns of all CBC intermediates can be formulated as a single constrained regression problem, avoiding the need for repeated simulation of time-resolved labeling patterns.
Results We then compare the flux estimates of the simulation-free constrained regression approach with those obtained from the classical INST-MFA based on labeling patterns of metabolites from the microalgae Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii under different growth conditions.
Discussion Our findings indicate that, in data-rich scenarios, simulation-free regression-based approaches provide a suitable alternative for flux estimation from classical INST-MFA since we observe a high qualitative agreement (rs=0.89) to predictions obtained from INCA, a state-of-the-art tool for INST-MFA.
The European Union is highly dependent on soybean imports from overseas to meet its protein demands. Individual Member States have been quick to declare self-sufficiency targets for plant-based proteins, but detailed strategies are still lacking. Rising global temperatures have painted an image of a bright future for soybean production in Europe, but emerging climatic risks such as drought have so far not been included in any of those outlooks.
Here, we present simulations of future soybean production and the most prominent risk factors across Europe using an ensemble of climate and soybean growth models. Projections suggest a substantial increase in potential soybean production area and productivity in Central Europe, while southern European production would become increasingly dependent on supplementary irrigation. Average productivity would rise by 8.3% (RCP 4.5) to 8.7% (RCP 8.5) as a result of improved growing conditions (plant physiology benefiting from rising temperature and CO2 levels) and farmers adapting to them by using cultivars with longer phenological cycles. Suitable production area would rise by 31.4% (RCP 4.5) to 37.7% (RCP 8.5) by the mid-century, contributing considerably more than productivity increase to the production potential for closing the protein gap in Europe.
While wet conditions at harvest and incidental cold spells are the current key challenges for extending soybean production, the models and climate data analysis anticipate that drought and heat will become the dominant limitations in the future. Breeding for heat-tolerant and water-efficient genotypes is needed to further improve soybean adaptation to changing climatic conditions.
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.
The physiological dependence of animals on dietary intake of vitamins, amino acids, and fatty acids is ubiquitous. Sharp differences in the availability of these vital dietary biomolecules among different resources mean that consumers must adopt a range of strategies to meet their physiological needs. We review the emerging work on omega-3 long-chain polyunsaturated fatty acids, focusing predominantly on predator-prey interactions, to illustrate that trade-off between capacities to consume resources rich in vital biomolecules and internal synthesis capacity drives differences in phenotype and fitness of consumers. This can then feedback to impact ecosystem functioning. We outline how focus on vital dietary biomolecules in eco-eco-devo dynamics can improve our understanding of anthropogenic changes across multiple levels of biological organization.
The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.
Cell-level systems biology model to study inflammatory bowel diseases and their treatment options
(2023)
To help understand the complex and therapeutically challenging inflammatory bowel diseases (IBDs), we developed a systems biology model of the intestinal immune system that is able to describe main aspects of IBD and different treatment modalities thereof. The model, including key cell types and processes of the mucosal immune response, compiles a large amount of isolated experimental findings from literature into a larger context and allows for simulations of different inflammation scenarios based on the underlying data and assumptions. In the context of a large and diverse virtual IBD population, we characterized the patients based on their phenotype (in contrast to healthy individuals, they developed persistent inflammation after a trigger event) rather than on a priori assumptions on parameter differences to a healthy individual. This allowed to reproduce the enormous diversity of predispositions known to lead to IBD. Analyzing different treatment effects, the model provides insight into characteristics of individual drug therapy. We illustrate for anti-TNF-alpha therapy, how the model can be used (i) to decide for alternative treatments with best prospects in the case of nonresponse, and (ii) to identify promising combination therapies with other available treatment options.
Leaves exhibit cells with varying degrees of shape complexity along the proximodistal axis. Heterogeneities in growth directions within individual cells bring about such complexity in cell shape. Highly complex and interconnected gene regulatory networks and signaling pathways have been identified to govern these processes. In addition, the organization of cytoskeletal networks and cell wall mechanical properties greatly influences the regulation of cell shape. Research has shown that microtubules are involved in regulating cellulose deposition and direc-tion of cell growth. However, comprehensive analysis of the regulation of the actin cytoskele-ton in cell shape regulation has not been well studied.
This thesis provides evidence that actin regulates aspects of cell growth, division, and direction-al expansion that impacts morphogenesis of developing leaves. The jigsaw puzzle piece mor-phology of epidermal pavement cells further serves as an ideal system to investigate the com-plex process of morphogenetic processes occurring at the cellular level. Here we have em-ployed live cell based imaging studies to track the development of pavement cells in actin com-promised conditions. Genetic perturbation of two predominantly expressed vegetative actin genes ACTIN2 and ACTIN7 results in delayed emergence of the cellular protrusions in pave-ment cells. Perturbation of actin also impacted the organization of microtubule in these cells that is known to promote emergence of cellular protrusions. Further, live-cell imaging of actin or-ganization revealed a correlation with cell shape, suggesting that actin plays a role in influencing pavement cell morphogenesis.
In addition, disruption of actin leads to an increase in cell size along the leaf midrib, with cells being highly anisotropic due to reduced cell division. The reduction of cell division further im-pacted the morphology of the entire leaf, with the mutant leaves being more curved. These re-sults suggests that actin plays a pivotal role in regulating morphogenesis at the cellular and tis-sue scales thereby providing valuable insights into the role of the actin cytoskeleton in plant morphogenesis.
The use of automated tools to reconstruct lipid metabolic pathways is not warranted in plants. Here, the authors construct Plant Lipid Module for Arabidopsis rosette using constraint-based modeling, demonstrate its integration in other plant metabolic models, and use it to dissect the genetic architecture of lipid metabolism.
Lipids play fundamental roles in regulating agronomically important traits. Advances in plant lipid metabolism have until recently largely been based on reductionist approaches, although modulation of its components can have system-wide effects. However, existing models of plant lipid metabolism provide lumped representations, hindering detailed study of component modulation. Here, we present the Plant Lipid Module (PLM) which provides a mechanistic description of lipid metabolism in the Arabidopsis thaliana rosette. We demonstrate that the PLM can be readily integrated in models of A. thaliana Col-0 metabolism, yielding accurate predictions (83%) of single lethal knock-outs and 75% concordance between measured transcript and predicted flux changes under extended darkness. Genome-wide associations with fluxes obtained by integrating the PLM in diel condition- and accession-specific models identify up to 65 candidate genes modulating A. thaliana lipid metabolism. Using mutant lines, we validate up to 40% of the candidates, paving the way for identification of metabolic gene function based on models capturing natural variability in metabolism.
Background
Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high-quality genome assembly and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular.
Results
A high-quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862 Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A Computational Analysis of gene Family Evolution (CAFE5) comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris.
Conclusions
We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolution, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.
Progressive habitat fragmentation threatens plant species with narrow habitat requirements. While local environmental conditions define population growth rates and recruitment success at the patch level, dispersal is critical for population viability at the landscape scale. Identifying the dynamics of plant meta-populations is often confounded by the uncertainty about soil-stored population compartments. We combined a landscape-scale assessment of an amphibious plant's population structure with measurements of dispersal complexity in time to track dispersal and putative shifts in functional connectivity. Using 13 microsatellite markers, we analyzed the genetic structure of extant Oenanthe aquatica populations and their soil seed banks in a kettle hole system to uncover hidden connectivity among populations in time and space. Considerable spatial genetic structure and isolation-by-distance suggest limited gene flow between sites. Spatial isolation and patch size showed minor effects on genetic diversity. Genetic similarity found among extant populations and their seed banks suggests increased local recruitment, despite some evidence of migration and recent colonization. Results indicate stepping-stone dispersal across adjacent populations. Among permanent and ephemeral demes the resulting meta-population demography could be determined by source-sink dynamics. Overall, these spatiotemporal connectivity patterns support mainland-island dynamics in our system, highlighting the importance of persistent seed banks as enduring sources of genetic diversity.
Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.
Spatial and temporal variation in perceived predation risk is an important determinant of movement and foraging activity of animals. Foraging in this landscape of fear, individuals need to decide where and when to move, and what resources to choose. Foraging theory predicts the outcome of these decisions based on energetic trade-offs, but complex interactions between perceived predation risk and preferences of foragers for certain functional traits of their resources are rarely considered. Here, we studied the interactive effects of perceived predation risk on food trait preferences and foraging behavior in bank voles (Myodes glareolus) in experimental landscapes. Individuals (n = 19) were subjected for periods of 24 h to two extreme, risk-uniform landscapes (either risky or safe), containing 25 discrete food patches, filled with seeds of four plant species in even amounts. Seeds varied in functional traits: size, nutrients, and shape. We evaluated whether and how risk modifies forager preference for functional traits. We also investigated whether perceived risk and distance from shelter affected giving-up density (GUD), time in patches, and number of patch visits. In safe landscapes, individuals increased time spent in patches, lowered GUD and visited distant patches more often compared to risky landscapes. Individuals preferred bigger seeds independent of risk, but in the safe treatment they preferred fat-rich over carb-rich seeds. Thus, higher densities of resource levels remained in risky landscapes, while in safe landscapes resource density was lower and less diverse due to selective foraging. Our results suggest that the interaction of perceived risk and dietary preference adds an additional layer to the cascading effects of a landscape of fear which affects biodiversity at resource level.
Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N & B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.
The oil palm (Elaeis guineensis Jacq.) produces a large amount of oil from the fruit. However, increasing the oil production in this fruit is still challenging. A recent study has shown that starch metabolism is essential for oil synthesis in fruit-producing species. Therefore, the transcriptomic analysis by RNA-seq was performed to observe gene expression alteration related to starch metabolism genes throughout the maturity stages of oil palm fruit with different oil yields. Gene expression profiles were examined with three different oil yields group (low, medium, and high) at six fruit development phases (4, 8, 12, 16, 20, and 22 weeks after pollination). We successfully identified and analyzed differentially expressed genes in oil palm mesocarps during development. The results showed that the transcriptome profile for each developmental phase was unique. Sucrose flux to the mesocarp tissue, rapid starch turnover, and high glycolytic activity have been identified as critical factors for oil production in oil palms. For starch metabolism and the glycolytic pathway, we identified specific gene expressions of enzyme isoforms (isozymes) that correlated with oil production, which may determine the oil content. This study provides valuable information for creating new high-oil-yielding palm varieties via breeding programs or genome editing approaches.
Keeping cool on hot days
(2023)
Long-lived organisms are likely to respond to a rapidly changing climate with behavioral flexibility. Animals inhabiting the arid parts of southern Africa face a particularly rapid rise in temperature which in combination with food and water scarcity places substantial constraints on the ability of animals to tolerate heat. We investigated how three species of African antelope-springbok Antidorcas marsupialis, kudu Tragelaphus strepsiceros and eland T. oryx-differing in body size, habitat preference and movement ecology, change their activity in response to extreme heat in an arid savanna. Serving as a proxy for activity, dynamic body acceleration data recorded every five minutes were analyzed for seven to eight individuals per species for the three hottest months of the year. Activity responses to heat during the hottest time of day (the afternoons) were investigated and diel activity patterns were compared between hot and cool days. Springbok, which prefer open habitat, are highly mobile and the smallest of the species studied, showed the greatest decrease in activity with rising temperature. Furthermore, springbok showed reduced mean activity over the 24 h cycle on hot days compared to cool days. Large-bodied eland seemed less affected by afternoon heat than springbok. While eland also reduced diurnal activity on hot days compared to cool days, they compensated for this by increasing nocturnal activity, possibly because their predation risk is lower. Kudu, which are comparatively sedentary and typically occupy shady habitat, seemed least affected during the hottest time of day and showed no appreciable difference in diel activity patterns between hot and cool days. The interplay between habitat preference, body size, movement patterns, and other factors seems complex and even sub-lethal levels of heat stress have been shown to impact an animal's long-term survival and reproduction. Thus, differing heat tolerances among species could result in a shift in the composition of African herbivore communities as temperatures continue to rise, with significant implications for economically important wildlife-based land use and conservation.
Starch has been a convenient, economically important polymer with substantial applications in the food and processing industry. However, native starches present restricted applications, which hinder their industrial usage. Therefore, modification of starch is carried out to augment the positive characteristics and eliminate the limitations of the native starches. Modifications of starch can result in generating novel polymers with numerous functional and value-added properties that suit the needs of the industry. Here, we summarize the possible starch modifications in planta and outside the plant system (physical, chemical, and enzymatic) and their corresponding applications. In addition, this review will highlight the implications of each starch property adjustment.
Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis
(2023)
Background
Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq.
Results
HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes.
Conclusions
Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.
The heat is on
(2023)
Climate conditions severely impact the activity and, consequently, the fitness of wildlife species across the globe. Wildlife can respond to new climatic conditions, but the pace of human-induced change limits opportunities for adaptation or migration. Thus, how these changes affect behavior, movement patterns, and activity levels remains unclear. In this study, we investigate how extreme weather conditions affect the activity of European hares (Lepus europaeus) during their peak reproduction period. When hares must additionally invest energy in mating, prevailing against competitors, or lactating, we investigated their sensitivities to rising temperatures, wind speed, and humidity. To quantify their activity, we used the overall dynamic body acceleration (ODBA) calculated from tri-axial acceleration measurements of 33 GPS-collared hares. Our analysis revealed that temperature, humidity, and wind speed are important in explaining changes in activity, with a strong response for high temperatures above 25 & DEG;C and the highest change in activity during temperature extremes of over 35 & DEG;C during their inactive period. Further, we found a non-linear relationship between temperature and activity and an interaction of activity changes between day and night. Activity increased at higher temperatures during the inactive period (day) and decreased during the active period (night). This decrease was strongest during hot tropical nights. At a stage of life when mammals such as hares must substantially invest in reproduction, the sensitivity of females to extreme temperatures was particularly pronounced. Similarly, both sexes increased their activity at high humidity levels during the day and low wind speeds, irrespective of the time of day, while the effect of humidity was stronger for males. Our findings highlight the importance of understanding the complex relationships between extreme weather conditions and mammal behavior, critical for conservation and management. With ongoing climate change, extreme weather events such as heat waves and heavy rainfall are predicted to occur more often and last longer. These events will directly impact the fitness of hares and other wildlife species and hence the population dynamics of already declining populations across Europe.
Light is the essential energy source for plants to drive photosynthesis. In nature, light availability is highly variable and often fluctuates on very short time scales. As a result, plants developed mechanisms to cope with these fluctuations. Understanding how to improve light use efficiency in natural fluctuating light (FL) conditions is a major target for agronomy.
In the first project, we identified an Arabidopsis thaliana plant that showed reduced levels of rapidly inducible non-photochemical quenching (NPQ). This plant was devoid of any T-DNA insertion. Using a mapping-by-sequencing approach, we successfully located the causal genomic region near the end of chromosome 4. Through variant investigations in that region, we identified a deletion of about 20 kb encompassing 9 genes. By complementation analysis, we confirmed that one of the deleted genes, VTC2, is the causal gene responsible for the low NPQ. Loss of VTC2 decreased NPQ particularly in old leaves, with young leaves being only slightly affected. Additionally, ascorbate levels were almost abolished in old leaves, likely causing the NPQ decrease by reducing the activity of the xanthophyll cycle. Although ascorbate levels in younger leaves were reduced compared to wild-type plants, they remained at a comparably higher level. This difference may be due to the VTC2 paralog VTC5, which is expressed at a higher level in young leaves than in old ones.
Plants require the PROTON GRADIENT REGULATION 5 (PGR5) protein for survival in FL. pgr5 mutants die because they fail to increase the luminal proton concentration in response to high light (HL) phases. A rapid elevation in ∆pH is needed to slow down electron transport through the Cytochrome b6 f complex (photosynthetic control). In FL, such lack of control in the pgr5 mutants results in photosystem I (PSI) overreduction, reactive oxygen species (ROS) production, and cell death. Decreases in photosystem II (PSII) activity introduced by crossing pgr5 with PSII deficient mutants
rescued the lethality of pgr5 in FL. PGR5 was suggested to act as part of the ferredoxin-plastoquinone reductase (FQR), involved in cyclic electron transfer around PSI. However, the proposed molecular role of PGR5 remains highly debated. To learn more about PGR5 function, we performed a forward genetic screen in Arabidopsis thaliana to identify EMS-induced suppressor mutants surviving longer when grown in FL compared to pgr5 mutants (referred to as ”suppressor of pgr5 lethality in fluctuating light”, splf ). 11 different candidate genes were identified in a total of 22 splf plants.
Mutants of seven of these genes in the pgr5 background showed low Fv/Fm values when grown in non-fluctuating low light (LL). Five of these 4genes were previously reported to have a role in PSII biogenesis or function. Two others, RPH1 and a DEAD/DEAH box helicase (AT3G02060), have not been linked to PSII function before. Three of splf candidate genes link to primary metabolism, fructose-2,6-bisphosphatase (F2KP ), udp-glucose pyrophosphorylase 1 (UGP1 ) and ferredoxin-dependent glutamate synthase (Fd-GOGAT ). They are characterized by the fact that they survive longer in FL than pgr5 mutants but do not procede beyond the early vegetative
phase and then die.
Plant metabolism serves as the primary mechanism for converting assimilated carbon into essential compounds crucial for plant growth and ultimately, crop yield. This renders it a focal point of research with significant implications. Despite notable strides in comprehending the genetic principles underpinning metabolism and yield, there remains a dearth of knowledge regarding the genetic factors responsible for trait variation under varying environmental conditions. Given the burgeoning global population and the advancing challenges posed by climate change, unraveling the intricacies of metabolic and yield responses to water scarcity became increasingly important in safeguarding food security.
Our research group has recently started to work on the genetic resources of legume species. To this end, the study presented here investigates the metabolic diversity across five different legume species at a tissue level, identifying species-specific biosynthesis of alkaloids as well as iso-/flavonoids with diverse functional groups, namely prenylation, phenylacylation as well as methoxylation, to create a resource for follow up studies investigation the metabolic diversity in natural diverse populations of legume species.
Following this, the second study investigates the genetic architecture of drought-induced changes in a global common bean population. Here, a plethora of quantitative trait loci (QTL) associated with various traits are identified by performing genome-wide association studies (GWAS), including for lipid signaling. On this site, overexpression of candidates highlighted the induction of several oxylipins reported to be pivotal in coping with harsh environmental conditions such as water scarcity.
Diverging from the common bean and GWAS, the following study focuses on identifying drought-related QTL in tomato using a bi-parental breeding population. This descriptive study highlights novel multi-omic QTL, including metabolism, photosynthesis as well as fruit setting, some of which are uniquely assigned under drought. Compared to conventional approaches using the bi-parental IL population, the study presented improves the resolution by assessing further backcrossed ILs, named sub-ILs.
In the final study, a photosynthetic gene, namely a PetM subunit of the cytochrome b6f complex encoding gene, involved in electron flow is characterized in an horticultural important crop. While several advances have been made in model organisms, this study highlights the transition of this fundamental knowledge to horticultural important crops, such as tomato, and investigates its function under differing light conditions. Overall, the presented thesis combines different strategies in unveiling the genetic components in multi-omic traits under drought using conventional breeding populations as well as a diverse global population. To this end, it allows a comparison of either approach and highlights their strengths and weaknesses.
Animal movement is a crucial aspect of life, influencing ecological and evolutionary processes. It plays an important role in shaping biodiversity patterns, connecting habitats and ecosystems. Anthropogenic landscape changes, such as in agricultural environments, can impede the movement of animals by affecting their ability to locate resources during recurring movements within home ranges and, on a larger scale, disrupt migration or dispersal. Inevitably, these changes in movement behavior have far-reaching consequences on the mobile link functions provided by species inhabiting such extensively altered matrix areas. In this thesis, I investigate the movement characteristics and activity patterns of the European hare (Lepus europaeus), aiming to understand their significance as a pivotal species in fragmented agricultural landscapes. I reveal intriguing results that shed light on the importance of hares for seed dispersal, the influence of personality traits on behavior and space use, the sensitivity of hares to extreme weather conditions, and the impacts of GPS collaring on mammals' activity patterns and movement behavior.
In Chapter I, I conducted a controlled feeding experiment to investigate the potential impact of hares on seed dispersal. By additionally utilizing GPS data of hares in two contrasting landscapes, I demonstrated that hares play a vital role, acting as effective mobile linkers for many plant species in small and isolated habitat patches. The analysis of seed intake and germination success revealed that distinct seed traits, such as density, surface area, and shape, profoundly affect hares' ability to disperse seeds through endozoochory. These findings highlight the interplay between hares and plant communities and thus provide valuable insights into seed dispersal mechanisms in fragmented landscapes.
By employing standardized behavioral tests in Chapter II, I revealed consistent behavioral responses among captive hares while simultaneously examining the intricate connection between personality traits and spatial patterns within wild hare populations. This analysis provides insights into the ecological interactions and dynamics within hare populations in agricultural habitats. Examining the concept of animal personality, I established a link between personality traits and hare behavior. I showed that boldness, measured through standardized tests, influences individual exploration styles, with shy and bold hares exhibiting distinct space use patterns. In addition to providing valuable insights into the role of animal personality in heterogeneous environments, my research introduced a novel approach demonstrating the feasibility of remotely assessing personality types using animal-borne sensors without additional disturbance of the focal individual.
While climate conditions severely impact the activity and, consequently, the fitness of wildlife species across the globe, in Chapter III, I uncovered the sensitivity of hares to temperature, humidity, and wind speed during their peak reproduction period. I found a strong response in activity to high temperatures above 25°C, with a particularly pronounced effect during temperature extremes of over 35°C. The non-linear relationship between temperature and activity was characterized by contrasting responses observed for day and night. These findings emphasize the vulnerability of hares to climate change and the potential consequences for their fitness and population dynamics with the ongoing rise of temperature.
Since such insights can only be obtained through capturing and tagging free-ranging animals, I assessed potential impacts and the recovery process post-collar attachment in Chapter IV. For this purpose, I examined the daily distances moved and the temporal-associated activity of 1451 terrestrial mammals out of 42 species during their initial tracking period. The disturbance intensity and the speed of recovery varied across species, with herbivores, females, and individuals captured and collared in relatively secluded study areas experiencing more pronounced disturbances due to limited anthropogenic influences.
Mobile linkers are essential for maintaining biodiversity as they influence the dynamics and resilience of ecosystems. Furthermore, their ability to move through fragmented landscapes makes them a key component for restoring disturbed sites. Individual movement decisions determine the scale of mobile links, and understanding variations in space use among individuals is crucial for interpreting their functions. Climate change poses further challenges, with wildlife species expected to adjust their behavior, especially in response to high-temperature extremes, and comprehending the anthropogenic influence on animal movements will remain paramount to effective land use planning and the development of successful conservation strategies.
This thesis provides a comprehensive ecological understanding of hares in agricultural landscapes. My research findings underscore the importance of hares as mobile linkers, the influence of personality traits on behavior and spatial patterns, the vulnerability of hares to extreme weather conditions, and the immediate consequences of collar attachment on mammalian movements. Thus, I contribute valuable insights to wildlife conservation and management efforts, aiding in developing strategies to mitigate the impact of environmental changes on hare populations. Moreover, these findings enable the development of methodologies aimed at minimizing the impacts of collaring while also identifying potential biases in the data, thereby benefiting both animal welfare and the scientific integrity of localization studies.
In this work, the role of the TusA protein was investigated for the cell functionality and FtsZ ring assembly in Escherichia coli. TusA is the tRNA-2-thiouridine synthase that acts as a sulfur transferase in tRNA thiolation for the formation of 2-thiouridine at the position 34 (wobble base) of tRNALys, tRNAGlu and tRNAGln. It binds the persulfide form of sulfur and transfers it to further proteins during mnm5s2U tRNA modification at wobble position and for Moco biosynthesis. With this thiomodification of tRNA, the ribosome binding is more efficient and frameshifting is averted during the protein translation. Previous studies have revealed an essential role of TusA in bacterial cell physiology since deletion of the tusA gene resulted in retarded growth and filamentous cells during the exponential growth phase in a rich medium which suddenly disappeared during the stationary phase. This indicates a problem in the cell division process. Therefore the focus of this work was to investigate the role of TusA for cell functionality and FtsZ ring formation and thus the cell separation.
The reason behind the filamentous growth of the tusA mutant strain was investigated by growth and morphological analyses. ΔtusA cells showed a retarded growth during the exponential phase compared to the WT strain. Also, morphological analysis of ΔtusA cells confirmed the filamentous cell shape. The growth and cell division defects in ΔtusA indicated a defect in FtsZ protein as a key player of cell division. The microscopic investigation revealed that filamentous ΔtusA cells possessed multiple DNA parts arranged next to each other. This suggested that although the DNA replication occurred correctly, there was a defect in the step where FtsZ should act; probably FtsZ is unable to assemble to the ring structure or the assembled ring is not able to constrict. All tested mutant strains (ΔtusD, ΔtusE and ΔmnmA) involved in the mnm5s2U34 tRNA modification pathway shared the similar retarded growth and filamentous cell shape like ΔtusA strain. Thus, the cell division defect arises from a defect in mnm5s2U34 tRNA thiolation.
Since the FtsZ ring formation was supposed to be defective in filaments, a possible intracellular interaction of TusA and FtsZ was examined by fluorescent (EGFP and mCherry) fusion proteins expression and FRET. FtsZ expressing tusA mutant (DE3) cells showed a red mCherry signal at the cell poles, indicating that FtsZ is still in the assembling phase. Interestingly, the cellular region of EGFP-TusA fusion protein expressed in ΔtusA (DE3) was conspicuous; the EGFP signal was spread throughout the whole cell and, in addition, a slight accumulation of the EGFP-TusA fluorescence was detectable at the cell poles, the same part of the cell as for mCherry-FtsZ. Thus, this strongly suggested an interaction of TusA and FtsZ.
Furthermore, the cellular FtsZ and Fis concentrations, and their change during different growth phases were determined via immunoblotting. All tested deletion strains of mnm5s2U34 tRNA modification show high cellular FtsZ and Fis levels in the exponential phase, shifting to the later growth phases. This shift reflects the retarded growth, whereby the deletion strains reach later the exponential phase. Conclusively, the growth and cell division defect, and thus the formation of filaments, is most likely caused by changes in the cellular FtsZ and Fis concentrations.
Finally, the translation efficiencies of certain proteins (RpoS, Fur, Fis and mFis) in tusA mutant and in additional gene deletion strains were studied whether they were affected by using unmodified U34 tRNAs of Lys, Glu and Gln. The translation efficiency is decreased in mnm5s2U34 tRNA modification-impaired strains in addition to their existing growth and cell division defect due to the elimination of these three amino acids. Finally, these results confirm and reinforce the importance of Lys, Glu and Gln and the mnm5s2U34 tRNA thiolation for efficient protein translation. Thus, these findings verify that the translation of fur, fis and rpoS is regulated by mnm5s2U34 tRNA modifications, which is growth phase-dependent.
In total, this work showed the importance of the role of TusA for bacterial cell functionality and physiology. The deletion of the tusA gene disrupted a complex regulatory network within the cell, that most influenced by the decreased translation of Fis and RpoS, caused by the absence of mnm5s2U34 tRNA modifications. The disruption of RpoS and Fis cellular network influences in turn the cellular FtsZ level in the early exponential phase. Finally, the reduced FtsZ concentration leads to elongated, filamentous E. coli cells, which are unable to divide.
The development of seeds in angiosperms starts with a complex process of double fertilization, involving the fusion of the maternal egg cell and central cell with two paternal sperm cells. This gives rise to the embryo and the nourishing endosperm, which are then enclosed by the seed coat, derived from the maternal integuments. The growth of the seed coat in Arabidopsis thaliana (Arabidopsis) is actively inhibited before fertilization by epigenetic regulators known as Polycomb Group (PcG) proteins. These proteins deposit a repressive histone mark called H3K27me3, which must be removed to enable seed coat formation. In this thesis, I explored the mechanism of removal of H3K27me3 marks from the integument cells following fertilization, which allows for seed coat formation. We hypothesized that this removal should be primarily facilitated by histone demethylases from the JMJ family and potentially influenced by the plant hormones Brassinosteroids (BRs). This hypothesis was supported by the expression patterns of the JMJ protein REF6 and of BR related genes, which are specifically expressed in the integuments and in the seed coat. Moreover, mutations in both these pathways lead to developmental defects, such as reduced ovule viability and delayed seed coat growth. Our research provides evidence suggesting that BR signalling is likely involved in recruiting JMJ-type histone demethylases to target loci responsible for seed coat growth. Moreover, we have discovered an additional pathway through which BRs regulate seed coat development, independent of their influence on H3K27me3 marks. This finding emphasizes the diverse roles of BRs in coordinating seed development, extending beyond their well-known involvement in plant growth and development. Furthermore, I explored the role of another epigenetic mark, DNA methylation, in fertilization-independent (or autonomous) seed formation in Arabidopsis. For this, we utilized epigenetic Recombinant Inbred Lines (epiRILs) and thus identified an epigenetic Quantitative Trait Locus (epiQTL) on chromosome II, potentially responsible for the larger autonomous seed size observed in DNA methylation mutants. Overall, this thesis significantly enhances our comprehension of the intricate relationship between epigenetic modifications, hormonal signaling, and plant reproductive processes. It offers valuable insights into the genetic mechanisms governing both sexual and asexual seed formation, while also presenting potential avenues for the engineer of advantageous traits in agricultural crops.
Die Fluoreszenz-Calcium-Imaging-Methode wird auch heute noch als gängige Methode verwendet, vor allem wegen der geringeren Kosten für das Wirkstoffscreening in der pharmazeutischen Forschung, wobei Ionenkanäle sowie einige der G-Protein gekoppelte Rezeptoren (GPCRs) die Mehrzahl der Wirkstoffziele ansprechen. Die zellfreie Synthese eukaryotischer Proteine hat nicht die Nachteile, die bei der Überexpression dieser ionenpermeablen Proteine in Zellen auftreten können, wie z. B. Zelltoxizität, geringere Proteinexpression und die Beseitigung der exprimierten Proteine aufgrund veränderter Domänen sowie die zeitaufwändige Pflege von Zelllinien. Die Synthese von Ionenkanälen in zellfreien Proteinsyntheseplattformen für das künftige Wirkstoffscreening ist noch in der Grundlagenforschung. Obwohl die Fluoreszenz-Calcium-Imaging-Methode in zellbasierten Assays weit verbreitet ist, wurde diese Methode bisher noch nicht in zellfreien Proteinexpressionssystemen verwendet. Insgesamt ist die neue Anwendung der Calcium-Imaging-Methode in eukaryontischen zellfreien Systemen eine Voraussetzung für die schnelle pharmakologische Analyse von Wirkstoffen. Das erste Ziel dieser wissenschaftlichen Arbeit bestand darin, die grundlegenden Prinzipien der Calcium-Imaging-Methode zur Untersuchung von Ionenkanälen in zellbasierten Systemen zu untersuchen. Hierfür wurden zwei Tumorzelllinien des Auges verwendet, und zwar benigne Pterygiumzellen und maligne Aderhautmelanom 92.1 Zellen. In diesen Studien wurde die Interaktion zwischen den nativ überexprimierten transient-receptor-potential-Ionenkanälen (TRPs) wie TRP Vanilliod 1 (TRPV1) (Capsaicinrezeptor) und TRP Melastatin 8 (TRPM8) (Mentholrezeptor) in diesen Tumorzellen nach Zugabe von verschiedenen Medikamenten und Hormonen untersucht. Das zweite Ziel dieser Arbeit war es, den Calcium-Mechanismus von GPCRs in den Zellen zu untersuchen. Zu diesem Zweck wurde Mas, ein GPCR und Angiotensin (1-7) -Hormonrezeptor, aus dem renin-angiotensin-aldosteron-system (RAAS) in der Human Embryonic Kidney-293 (HEK293) Zelllinie überexprimiert. In dieser Studie wurden insbesondere die Aktivierung klassischer GPCR-Signalwege wie Phospholipase C und Proteinkinase C durch Angiotensin-(1-7) über Mas und die Beteiligung von TRP-Kanälen nachgewiesen. Die zellbasierte-Calcium-Imaging-Methode für chemische Calcium-Indikatoren ließ sich aufgrund der Anwesenheit einer großen Menge cytosolischer Carboxylesterasen gut anwenden. Carboxylesterase ist das wichtigste Enzym in der Calcium Imaging Methode, das die Verarbeitung chemischen Calcium-Farbstoffe behandelt. Dieses Enzym fehlt jedoch in Mikrosomen, die als Basismembran für die Integration synthetisierter Ionenkanäle in eukaryontischen zellfreien Systemen verwendet werden. Das dritte Ziel dieser Forschungsarbeit war die Umsetzung der zellbasierten Calcium-Imaging Methode und der Calcium-Signalwege in zellfreie Systeme. Hier wurde die zellfrei synthetisierte Carboxylesterase in Mikrosomen von Spodoptera frugiperda (Sf21) als praktikables Calcium-Imaging-Werkzeug etabliert, um sowohl native ionenpermeable Proteine als auch zellfrei-synthetisierte Ionenkanäle zu untersuchen. Die Enzymaktivität der zellfrei-synthetisierten Carboxylesterase in Mikrosomen wurde durch Esterase-Assays und den Calcium-Fluoreszenzfarbstoff Fluo-5N Acetoxymethylester (Fluo-5N AM) Belastungstests nachgewiesen. Das Calcium-Imaging der nativ vorhandenen Ca2+-ATPase des sarkoplasmatischen/endoplasmatischen Retikulums (SERCA) und der Ryanodin-Rezeptoren (RyR) in den Mikrosomen sowie der zell-frei exprimierten TRP-Ionenkanäle wurden mit dem Fura-5N-AM- Fluoreszenzfarbstoff in mit Carboxylesterase vorsynthetisierten Mikrosomen nachgewiesen.
Zusammenfassend lässt sich sagen, dass das Prinzip der zellbasierten Calcium-Imaging -Methode vielversprechend an das eukaryotische zellfreie Sf21-System angepasst werden konnte, um Ionenkanäle zu analysieren. Nach entsprechender Forschung könnte die etablierte Methode in Zukunft auch auf andere Membranproteine ausgeweitet werden. Dies umfasst die Untersuchung anderer zell-frei exprimierte GPCRs oder anderer Ionenkanäle wie Kalium-, Natrium- und Chlorid-Ionenkanäle.
Conservation of the jaguar relies on holistic and transdisciplinary conservation strategies that integratively safeguard essential, connected habitats, sustain viable populations and their genetic exchange, and foster peaceful human-jaguar coexistence. These strategies define four research priorities to advance jaguar conservation throughout the species’ range. In this thesis I provide several relevant ecological and sociological insights into these research priorities, each addressed in a separate chapter. I focus on the effects of anthropogenic landscapes on jaguar habitat use and population gene flow, spatial patterns of jaguar habitat suitability and functional population connectivity, and on innovative governance approaches which can work synergistically to help achieve human-wildlife conviviality. Furthermore, I translate these insights into recommendations for conservation practice by providing tools and suggestions that conservation managers and stakeholders can use to implement local actions but also make broad scale conservation decisions in Central America. In Chapter 2, I model regional habitat use of jaguars, producing spatially-explicit maps for management of key areas of habitat suitability. Using an occupancy model of 13-year-camera-trap occurrence data, I show that human influence has the strongest impact on jaguar habitat use, and that Jaguar Conservation Units are the most important reservoirs of high quality habitat in this region. I build upon these results by zooming in to an area of high habitat suitability loss in Chapter 3, northern Central America. Here I study the drivers of jaguar gene flow and I produce spatially-explicit maps for management of key areas of functional population connectivity in this region. I use microsatellite data and pseudo-optimized multiscale, multivariate resistance surfaces of gene flow to show that jaguar gene flow is influenced by environmental, and even more strongly, by human influence variables; and that the areas of lowest gene flow resistance largely coincide with the location of the Jaguar Conservation Units. Given that human activities significantly impact jaguar habitat use and gene flow, securing viable jaguar populations in anthropogenic landscapes also requires fostering peaceful human-wildlife coexistence. This is a complex challenge that cannot be met without transdisciplinary academic research and cross-sectoral, collaborative governance structures that effectively respond to the multiple challenges of such coexistence. With this in mind, I focus in Chapter 4 on carnivore conservation initiatives that apply transformative governance approaches to enact transformative change towards human-carnivore coexistence. Using the frameworks of transformative biodiversity governance and convivial conservation, I highlight in this chapter concrete pathways, supported by more inclusive, democratic forms of conservation decision-making and participation that promote truly transformative changes towards human-jaguar conviviality.
Starch is a biopolymer for which, despite its simple composition, understanding the precise mechanism behind its formation and regulation has been challenging. Several approaches and bioanalytical tools can be used to expand the knowledge on the different parts involved in the starch metabolism. In this sense, a comprehensive analysis targeting two of the main groups of molecules involved in this process: proteins, as effectors/regulators of the starch metabolism, and maltodextrins as starch components and degradation products, was conducted in this research work using potato plants (Solanum tuberosum L. cv. Desiree) as model of study. On one side, proteins physically interacting to potato starch were isolated and analyzed through mass spectrometry and western blot for their identification. Alternatively, starch interacting proteins were explored in potato tubers from transgenic plants having antisense inhibition of starch-related enzymes and on tubers stored under variable environmental conditions. Most of the proteins recovered from the starch granules corresponded to previously described proteins having a specific role in the starch metabolic pathway. Another set of proteins could be grouped as protease inhibitors, which were found weakly interacting to starch. Variations in the protein profile obtained after electrophoresis separation became clear when tubers were stored under different temperatures, indicating a differential expression of proteins in response to changing environmental conditions.
On the other side, since maltodextrin metabolism is thought to be involved in both starch initiation and degradation, soluble maltooligosaccharide content in potato tubers was analyzed in this work under diverse experimental variables. For this, tuber disc samples from wild type and transgenic lines strongly repressing either the plastidial or cytosolic form of the -glucan phosphorylase and phosphoglucomutase were incubated with glucose, glucose-6-phosphate, and glucose-1-phosphate solutions to evaluate the influence of such enzymes on the conversion of the carbon sources into soluble maltodextrins, in comparison to wild-type samples. Relative maltodextrin amounts analyzed through capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF) revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30 (DP30), in contrast to transgenic tubers with strong repression of the plastidial glucan phosphorylase. The results obtained from the maltodextrin analysis support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucan polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial glucan phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.
In the present thesis, AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, were demonstrated as a simple and fast method to functionalize the surface of nanoelectrodes with submicrometer sized biological objects. These nanoelectrodes have a cylindrical shape with a diameter of 500 nm arranged in an array of 6256 electrodes. Due to its medical relevance influenza virus as well as anti-influenza antibodies were chosen as a model organism. Common methods to bring antibodies or proteins to biosensor surfaces are complex and time-consuming. In the present work, it was demonstrated that by applying AC electric fields influenza viruses and antibodies can be immobilized onto the nanoelectrodes within seconds without any prior chemical modification of neither the surface nor the immobilized biological object. The distribution of these immobilized objects is not uniform over the entire array, it exhibits a decreasing gradient from the outer row to the inner ones. Different causes for this gradient have been discussed, such as the vortex-shaped fluid motion above the nanoelectrodes generated by, among others, electrothermal fluid flow. It was demonstrated that parts of the accumulated material are permanently immobilized to the electrodes. This is a unique characteristic of the presented system since in the literature the AC electrokinetic immobilization is almost entirely presented as a method just for temporary immobilization. The spatial distribution of the immobilized viral material or the anti-influenza antibodies at the electrodes was observed by either the combination of fluorescence microscopy and deconvolution or by super-resolution microscopy (STED). On-chip immunoassays were performed to examine the suitability of the functionalized electrodes as a potential affinity-based biosensor. Two approaches were pursued: A) the influenza virus as the bio-receptor or B) the influenza virus as the analyte. Different sources of error were eliminated by ELISA and passivation experiments. Hence, the activity of the immobilized object was inspected by incubation with the analyte. This resulted in the successful detection of anti-influenza antibodies by the immobilized viral material. On the other hand, a detection of influenza virus particles by the immobilized anti-influenza antibodies was not possible. The latter might be due to lost activity or wrong orientation of the antibodies. Thus, further examinations on the activity of by AC electric fields immobilized antibodies should follow. When combined with microfluidics and an electrical read-out system, the functionalized chips possess the potential to serve as a rapid, portable, and cost-effective point-of-care (POC) device. This device can be utilized as a basis for diverse applications in diagnosing and treating influenza, as well as various other pathogens.
Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
Im Rahmen des PSI-Projekts wurde eine Lehrveranstaltung konzipiert, die Lehramtsstudierenden einen vertieften Einblick sowohl in den Ablauf von Forschung als auch eine Bearbeitung einer eigenen experimentellen Forschungsaufgabe ermöglichen soll. Anlass waren die Berücksichtigung eines „Wissens über Erkenntnisgewinnung in der Disziplin“ im Modell des „Erweiterten Fachwissens für den schulischen Kontext“ (PSI) sowie Erkenntnisse empirischer Studien, die die Relevanz eigener Forschungserfahrung für das Unterrichten naturwissenschaftlicher Erkenntnisgewinnungsprozesse zeigen. Hier stellen wir eine neue Lehrveranstaltung (4 SWS) vor, die den angehenden Lehrkräften Forschungserfahrung ermöglicht (Seminar und Praktikum). Die Lehrveranstaltung vermittelt Einblicke in Forschung und die „Natur der Naturwissenschaften“, ermöglicht das Durchführen eigener wissenschaftlicher und schulrelevanter Experimente und bietet eine angemessene Reflexion über die verschiedenen Kurselemente. Die Evaluationsergebnisse sind überwiegend positiv, zeigen aber auch, dass für die Studierenden die wahrgenommene Schulrelevanz und die fachdidaktischen Aspekte ein wichtiges Kriterium für die positive Bewertung sind.
Dielektrophorese ist die Manipulation polarisierbarer Partikel durch inhomogene elektrische Wechselfelder. In dieser Arbeit wurden drei verschiedene Enzyme durch Dielektrophorese immobilisiert und anschließend hinsichtlich ihrer katalytischen Aktivität untersucht: Meerrettichperoxidase, Cholinoxidase aus Alcaligenes sp. und Glucoseoxidase aus Aspergillus niger. Die Immobilisierung erfolgte durch Dielektrophorese auf nano-Elektrodenarrays aus Wolfram-Zylindern mit 500 nm Durchmesser oder aus Titannitrid-Ringen mit 20 nm Breite. Die Immobilisierung der Enzyme konnte fluoreszenzmikroskopisch entweder anhand der intrinsischen Fluoreszenz oder aufgrund einer Fluoreszenzmarkierung vor oder nach der Immobilisierung für alle getesteten Enzyme nachgewiesen werden. Die Messung der Enzymaktivität erfolgte quantitativ durch den direkten oder indirekten Nachweis des gebildeten Produktes oder, im Falle der Cholinoxidase, durch Beobachtung der intrinsischen Fluoreszenz des Cofaktors FAD, die vom Oxidationszustand dieses Enzyms abhängt. Für die Meerrettichperoxidase konnte so eine hohe erhaltene Enzymaktivität nach der Immobilisierung nachgewiesen werden. Die Aktivität der permanent immobilisierten Fraktion der Meerrettichperoxidase entsprach bis zu 47 % der höchstmöglichen Aktivität einer Monolage dieses Enzyms auf den Elektroden des Chips. Diese Aktivität kann als aktive, aber zufällig gegenüber der Oberfläche ausgerichtete Enzymschicht interpretiert werden. Für die permanent immobilisierte Glucoseoxidase wurde nur eine Aktivität entsprechend <1,3 % der Aktivität einer solchen Enzymschicht detektiert, während für die immobilisierte Cholinoxidase gar keine Aktivität nachgewiesen werden konnte. Die Aktivität der durch DEP immobilisierten Enzyme konnte somit quantitativ bestimmt werden. Der Anteil an erhaltener Aktivität hängt dabei stark vom verwendeten Enzym ab.
Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana
(2023)
The emerging threat of antibiotic-resistant bacteria has become a global challenge in the last decades, leading to a rising demand for alternative treatments for bacterial infections. One approach is to target the bacterial cell envelope, making understanding its biophysical properties crucial. Specifically, bacteriophages use the bacterial envelope as an entry point to initiate infection, and they are considered important building blocks of new antibiotic strategies against drug-resistant bacteria.. Depending on the structure of the cell wall, bacteria are classified as Gram-negative and Gram-positive. Gram-negative bacteria are equipped with a complex cell envelope composed of two lipid membranes enclosing a rigid peptidoglycan layer. The synthesis machinery of the Gram-negative cell envelope is the target of antimicrobial agents, including new physical sanitizing procedures addressing the outer membrane (OM). It is therefore very important to study the biophysical properties of the Gram-negative bacterial cell envelope. The high complexity of the Gram-negative OM sets the demand for a model system in which the contribution of individual components can be evaluated separately. In this respect, giant unilamellar vesicles (GUVs) are promising membrane systems to study membrane properties while controlling parameters such as membrane composition and surrounding medium conditions.
The aim of this work was to develop methods and approaches for the preparation and characterization of a GUV-based membrane model that mimics the OM of the Gram-negative cell envelope. A major component of the OM is the lipopolysaccharide (LPS) on the outside of the OM heterobilayer. The vesicle model was designed to contain LPS in the outer leaflet and lipids in the inner leaflet. Furthermore, the interaction of the prepared LPS-GUVs with bacteriophages was tested. LPS containing GUVs were prepared by adapting the inverted emulsion technique to meet the challenging properties of LPS, namely their high self-aggregation rate in aqueous solutions. Notably, an additional emulsification step together with the adaption of solution conditions was employed to asymmetrically incorporate LPS containing long polysaccharide chains into the artificial membranes. GUV membrane asymmetry was verified with a fluorescence quenching assay. Since the necessary precautions for handling the quenching agent sodium dithionite are often underestimated and poorly described, important parameters were tested and identified to obtain a stable and reproducible assay. In the context of varied LPS incorporation, a microscopy-based technique was introduced to determine the LPS content on individual GUVs and to directly compare vesicle properties and LPS coverage. Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.
Employing LPS-GUVs we could demonstrate that a Salmonella bacteriophage bound with high specificity to its LPS receptor when presented at the GUV surface, and that the number of bound bacteriophages scaled with the amount of presented LPS receptor. In addition to binding, the bacteriophages were able to eject their DNA into the vesicle lumen. LPS-GUVs thus provide a starting platform for bottom-up approaches for the generation of more complex membranes, in which the effects of individual components on the membrane properties and the interaction with antimicrobial agents such as bacteriophages could be explored.
Aptamers are single-stranded DNA (ssDNA) or RNA molecules that can bind specifically and with high affinity to target molecules due to their unique three-dimensional structure. For this reason, they are often compared to antibodies and sometimes even referred to as “chemical antibodies”. They are simple and inexpensive to synthesize, easy to modify, and smaller than conventional antibodies. Enzymes, especially hydrolases, are interesting targets in this context. This class of enzymes is capable of hydrolytically cleaving various macromolecules such as proteins, as well as smaller molecules such as antibiotics. Hence, they play an important role in many biological processes including diseases and their treatment. Hydrolase detection as well as the understanding of their function is therefore of great importance for diagnostics and therapy. Due to their various desirable features compared to antibodies, aptamers are being discussed as alternative agents for analytical and diagnostic use in various applications. The use of aptamers in therapy is also frequently investigated, as the binding of aptamers can have effects on the catalytic activity, protein-protein interactions, or proteolytic cascades. Aptamers are generated by an in vitro selection process. Potential aptamer candidates are selected from a pool of enriched nucleic acid sequences with affinity to the target, and their binding affinity and specificity is investigated. This is one of the most important steps in aptamer generation to obtain specific aptamers with high affinity for use in analytical and diagnostic applications. The binding properties or binding domains and their effects on enzyme functions form the basis for therapeutic applications.
In this work, the binding properties of DNA aptamers against two different hydrolases were investigated. In view of their potential utility for analytical methods, aptamers against human urokinase (uPA) and New Delhi metallo-β-lactamase-1 (NDM-1) were evaluated for their binding affinity and specificity using different methods. Using the uPA aptamers, a protocol for measuring the binding kinetics of an aptamer-protein-interaction by surface plasmon resonance spectroscopy (SPR) was developed. Based on the increased expression of uPA in different types of cancer, uPA is discussed as a prognostic and diagnostic tumor marker. As uPA aptamers showed different binding sites on the protein, microtiter plate-based aptamer sandwich assay systems for the detection of uPA were developed. Because of the function of urokinase in cancer cell proliferation and metastasis, uPA is also discussed as a therapeutic target. In this regard, the different binding sites of aptamers showed different effects on uPA function. In vitro experiments demonstrated both inhibition of uPA binding to its receptor as well as the inhibition of uPA catalytic activity for different aptamers. Thus, in addition to their specificity and affinity for their targets, the utility of the aptamers for potential diagnostic and therapeutic applications was demonstrated. First, as an alternative inhibitor of human urokinase for therapeutic purposes, and second, as valuable recognition molecules for the detection of urokinase, as a prognostic and diagnostic marker for cancer, and for NDM-1 to detect resistance to carbapenem antibiotics.
Inflammatory bowel diseases (IBD), characterised by a chronic inflammation of the gut wall, develop as consequence of an overreacting immune response to commensal bacteria, caused by a combination of genetic and environmental conditions. Large inter-individual differences in the outcome of currently available therapies complicate the decision for the best option for an individual patient. Predicting the prospects of therapeutic success for an individual patient is currently only possible to a limited extent; for this, a better understanding of possible differences between responders and non-responders is needed.
In this thesis, we have developed a mathematical model describing the most important processes of the gut mucosal immune system on the cellular level. The model is based on literature data, which were on the one hand used (qualitatively) to choose which cell types and processes to incorporate and to derive the model structure, and on the other hand (quantitatively) to derive the parameter values. Using ordinary differential equations, it describes the concentration-time course of neutrophils, macrophages, dendritic cells, T cells and bacteria, each subdivided into different cell types and activation states, in the lamina propria and mesenteric lymph nodes. We evaluate the model by means of simulations of the healthy immune response to salmonella infection and mucosal injury.
A virtual population includes IBD patients, which we define through their initially asymptomatic, but after a trigger chronically inflamed gut wall. We demonstrate the model's usefulness in different analyses: (i) The comparison of virtual IBD patients with virtual healthy individuals shows that the disease is elicited by many small or fewer large changes, and allows to make hypotheses about dispositions relevant for development of the disease. (ii) We simulate the effects of different therapeutic targets and make predictions about the therapeutic outcome based on the pre-treatment state. (iii) From the analysis of differences between virtual responders and non-responders, we derive hypotheses about reasons for the inter-individual variability in treatment outcome. (iv) For the example of anti-TNF-alpha therapy, we analyse, which alternative therapies are most promising in case of therapeutic failure, and which therapies are most suited for combination therapies: For drugs also directly targeting the cytokine levels or inhibiting the recruitment of innate immune cells, we predict a low probability of success when used as alternative treatment, but a large gain when used in a combination treatment. For drugs with direct effects on T cells, via modulation of the sphingosine-1-phosphate receptor or inhibition of T cell proliferation, we predict a considerably larger probability of success when used as alternative treatment, but only a small additional gain when used in a combination therapy.
Biomolecules such as proteins and lipids have vital roles in numerous cellular functions, including biomolecule transport, protein functions, cellular homeostasis and biomembrane integrity. Traditional biochemistry methods do not provide precise information about cellular biomolecule distribution and behavior under native environmental conditions since they are not transferable to live cell samples. Consequently, this can lead to inaccuracies in quantifying biomolecule interactions due to potential complexities arising from the heterogeneity of native biomembranes. To overcome these limitations, minimal invasive microscopic techniques, such as fluorescence fluctuation spectroscopy (FFS) in combination with fluorescence proteins (FPs) and fluorescence lipid analogs, have been developed. FFS techniques and membrane property sensors enable the quantification of various parameters, including concentration, dynamics, oligomerization, and interaction of biomolecules in live cell samples.
In this work, several FFS approaches and membrane property sensors were implemented and employed to examine biological processes of diverse context. Multi-color scanning fluorescence fluctuation spectroscopy (sFCS) was used the examine protein oligomerization, protein-protein interactions (PPIs) and protein dynamics at the cellular plasma membrane (PM). Additionally, two-color number and brightness (N&B) analysis was extended with the cross-correlation analysis in order to quantify hetero-interactions of proteins in the PM with very slow motion, which would not accessible with sFCS due strong initial photobleaching. Furthermore, two semi-automatic analysis pipelines were designed: spectral Förster resonance energy transfer (FRET) analysis to study changes in membrane charge at the inner leaflet of the PM, and spectral generalized polarization (GP) imaging and spectral phasor analysis to monitor changes in membrane fluidity and order.
An important parameter for studying PPIs is molecular brightness, which directly determines oligomerization and can be extracted from FFS data. However, FPs often display complex photophysical transitions, including dark states. Therefore, it is crucial to characterize FPs for their dark-states to ensure reliable oligomerization measurements. In this study, N&B and sFCS analysis were applied to determine photophysical properties of novel green FPs under different conditions (i.e., excitation power and pH) in living cells. The results showed that the new FPs, mGreenLantern (mGL) and Gamillus, exhibited the highest molecular brightness at the cost of lower photostability. The well-established monomeric enhanced green fluorescent protein (mEGFP) remained the best option to investigate PPIs at lower pH, while mGL was best suited for neutral pH, and Gamillus for high pH. These findings provide guidance for selecting an appropriate FP to quantify PPIs via FFS under different environmental conditions.
Next, several biophysical fluorescence microscopy approaches (i.e., sFCS, GP imaging, membrane charge FRET) were employed to monitor changes in lipid-lipid-packing in biomembranes in different biological context. Lipid metabolism in cancer cells is known to support rapid proliferation and metastasis. Therefore, targeting lipid synthesis or membrane integrity holds immense promise as an anticancer strategy. However, the mechanism of action of the novel agent erufosine (EPC3) on membrane stability is not fully under
stood. The present work revealed that EPC3 reduces lipid packing and composition as well as increased membrane fluidity and dynamic, hence, modifies lipid-lipid-interaction. These effects on membrane integrity were likely triggered by modulations in lipid metabolism and membrane organization. In the case of influenza A virus (IAV) infection, regulation of lipid metabolism is crucial for multiple steps in IAV replication and is related to the pathogenicity of IAV. Here, it is shown for the first time that IAV infection triggers a local enrichment of negatively charged lipids at the inner leaflet of the PM, which decreases membrane fluidity and dynamic, as well as increases lipid packing at the assembly site in living cells. This suggests that IAV alters lipid-lipid interactions and organization at the PM. Overall, this work highlights the potential of biophysical techniques as a screening platform for studying membrane properties in living cells at the single-cell level.
Finally, this study addressed remaining questions about the early stage of IAV assembly. The recruitment of matrix protein 1 (M1) and its interaction with other viral surface proteins, hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2), has been a subject of debate due to conflicting results. In this study, different FFS approaches were performed in transfected cells to investigate interactions between IAV proteins themselves and host factors at the PM. FFS measurements revealed that M2 interacts strongly with M1, leading to the translocation of M1 to the PM. This interaction likely took place along the non-canonical pathway, as evidenced by the detection of an interaction between M2 and the host factor LC3-II, leading to the recruitment of LC3-II to the PM. Moreover, weaker interaction was observed between HA and membrane-bound M1, and no interaction between NA and M1. Interestingly, higher oligomeric states of M1 were only detectable in infected cells. These results indicate that M2 initiates virion assembly by recruiting M1 to the PM, which may serve as a platform for further interactions with viral proteins and host factors.
Life on Earth is diverse and ranges from unicellular organisms to multicellular creatures like humans. Although there are theories about how these organisms might have evolved, we understand little about how ‘life’ started from molecules. Bottom-up synthetic biology aims to create minimal cells by combining different modules, such as compartmentalization, growth, division, and cellular communication.
All living cells have a membrane that separates them from the surrounding aqueous medium and helps to protect them. In addition, all eukaryotic cells have organelles that are enclosed by intracellular membranes. Each cellular membrane is primarily made of a lipid bilayer with membrane proteins. Lipids are amphiphilic molecules that assemble into molecular bilayers consisting of two leaflets. The hydrophobic chains of the lipids in the two leaflets face each other, and their hydrophilic headgroups face the aqueous surroundings. Giant unilamellar vesicles (GUVs) are model membrane systems that form large compartments with a size of many micrometers and enclosed by a single lipid bilayer. The size of GUVs is comparable to the size of cells, making them good membrane models which can be studied using an optical microscope. However, after the initial preparation, GUV membranes lack membrane proteins which have to be reconstituted into these membranes by subsequent preparation steps. Depending on the protein, it can be either attached via anchor lipids to one of the membrane leaflets or inserted into the lipid bilayer via its transmembrane domains.
The first step is to prepare the GUVs and then expose them to an exterior solution with proteins. Various protocols have been developed for the initial preparation of GUVs. For the second step, the GUVs can be exposed to a bulk solution of protein or can be trapped in a microfluidic device and then supplied with the protein solution. To minimize the amount of solution and for more precise measurements, I have designed a microfluidic device that has a main channel, and several dead-end side channels that are perpendicular to the main channel. The GUVs are trapped in the dead-end channels. This design exchanges the solution around the GUVs via diffusion from the main channel, thus shielding the GUVs from the flow within the main channel. This device has a small volume of just 2.5 μL, can be used without a pump and can be combined with a confocal microscope, enabling uninterrupted imaging of the GUVs during the experiments. I used this device for most of the experiments on GUVs that are discussed in this thesis.
In the first project of the thesis, a lipid mixture doped with an anchor lipid was used that can bind to a histidine chain (referred to as His-tag(ged) or 6H) via the metal cation Ni2+. This method is widely used for the biofunctionalization of GUVs by attaching proteins without a transmembrane domain. Fluorescently labeled His-tags which are bound to a membrane can be observed in a confocal microscope. Using the same lipid mixture, I prepared the GUVs with different protocols and investigated the membrane composition of the resulting GUVs by evaluating the amount of fluorescently labeled His-tagged molecules bound to their membranes. I used the microfluidic device described above to expose the outer leaflet of the vesicle to a constant concentration of the His-tagged molecules. Two fluorescent molecules with a His-tag were studied and compared: green fluorescent protein (6H-GFP) and fluorescein isothiocyanate (6H-FITC). Although the quantum yield in solution is similar for both molecules, the brightness of the membrane-bound 6H-GFP is higher than the brightness of the membrane-bound 6H-FITC. The observed difference in the brightness reveals that the fluorescence of the 6H-FITC is quenched by the anchor lipid via the Ni2+ ion. Furthermore, my measurements also showed that the fluorescence intensity of the membranebound His-tagged molecules depends on microenvironmental factors such as pH. For both 6H-GFP and 6H-FITC, the interaction with the membrane is quantified by evaluating the equilibrium dissociation constant. The membrane fluorescence is measured as a function of the fluorophores’ molar concentration. Theoretical analysis of these data leads to the equilibrium dissociation constants of (37.5 ± 7.5) nM for 6H-GFP and (18.5 ± 3.7) nM for 6H-FITC.
The anchor lipid mentioned previously used the metal cation Ni2+ to mediate the bond between the anchor lipid and the His-tag. The Ni2+ ion can be replaced by other transition metal ions. Studies have shown that Co3+ forms the strongest bonds with the His-tags attached to proteins. In these studies, strong oxidizing agents were used to oxidize the Co2+ mediated complex with the His-tagged protein to a Co3+ mediated complex. This procedure puts the proteins at risk of being oxidized as well. In this thesis, the vesicles were first prepared with anchor lipids without any metal cation. The Co3+ was added to these anchor lipids and finally the His-tagged protein was added to the GUVs to form the Co3+ mediated bond. This system was also established using the microfluidic device.
The different preparation procedures of GUVs usually lead to vesicles with a spherical morphology. On the other hand, many cell organelles have a more complex architecture with a non spherical topology. One fascinating example is provided by the endoplasmic reticulum (ER) which is made of a continuous membrane and extends throughout the cell in the form of tubes and sheets. The tubes are connected by three-way junctions and form a tubular network of irregular polygons. The formation and maintenance of these reticular networks requires membrane proteins that hydrolyize guanosine triphosphate (GTP). One of these membrane proteins is atlastin. In this thesis, I reconstituted the atlastin protein in GUV membranes using detergent-assisted reconstitution protocols to insert the proteins directly into lipid bilayers.
This thesis focuses on protein reconstitution by binding His-tagged proteins to anchor lipids and by detergent-assisted insertion of proteins with transmembrane domains. It also provides the design of a microfluidic device that can be used in various experiments, one example is the evaluation of the equilibrium dissociation constant for membrane-protein interactions. The results of this thesis will help other researchers to understand the protocols for preparing GUVs, to reconstitute proteins in GUVs, and to perform experiments using the microfluidic device. This knowledge should be beneficial for the long-term goal of combining the different modules of synthetic biology to make a minimal cell.
Sulfur is essential for the functionality of some important biomolecules in humans. Biomolecules like the Iron-sulfur clusters, tRNAs, Molybdenum cofactor, and some vitamins. The trafficking of sulfur involves proteins collectively called sulfurtransferase. Among these are TUM1, MOCS3, and NFS1.
This research investigated the role of TUM1 for molybdenum cofactor biosynthesis and cytosolic tRNA thiolation in humans. The rhodanese-like protein MOCS3 and the L-cysteine desulfurase (NFS1) have been previously demonstrated to interact with TUM1. These interactions suggested a dual function of TUM1 in sulfur transfer for Moco biosynthesis and cytosolic tRNA thiolation. TUM1 deficiency has been implicated to be responsible for a rare inheritable disorder known as mercaptolactate cysteine disulfiduria (MCDU), which is associated with a mental disorder. This mental disorder is similar to the symptoms of sulfite oxidase deficiency which is characterised by neurological disorders. Therefore, the role of TUM1 as a sulfurtransferase in humans was investigated, in CRISPR/Cas9 generated TUM1 knockout HEK 293T cell lines.
For the first time, TUM1 was implicated in Moco biosynthesis in humans by quantifying the intermediate product cPMP and Moco using HPLC. Comparing the TUM1 knockout cell lines to the wild-type, accumulation and reduction of cPMP and Moco were observed respectively. The effect of TUM1 knockout on the activity of a Moco-dependent enzyme, Sulfite oxidase, was also investigated. Sulfite oxidase is essential for the detoxification of sulfite to sulfate. Sulfite oxidase activity and protein abundance were reduced due to less availability of Moco. This shows that TUM1 is essential for efficient sulfur transfer for Moco biosynthesis. Reduction in cystathionin -lyase in TUM1 knockout cells was quantified, a possible coping mechanism of the cell against sulfite production through cysteine catabolism.
Secondly, the involvement of TUM1 in tRNA thio-modification at the wobble Uridine-34 was reported by quantifying the amount of mcm5s2U and mcm5U via HPLC. The reduction and accumulation of mcm5s2U and mcm5U in TUM1 knockout cells were observed in the nucleoside analysis. Herein, exogenous treatment with NaHS, a hydrogen sulfide donor, rescued the Moco biosynthesis, cytosolic tRNA thiolation, and cell proliferation deficits in TUM1 knockout cells.
Further, TUM1 was shown to impact mitochondria bioenergetics through the measurement of the oxygen consumption rate and extracellular acidification rate (ECAR) via the seahorse cell Mito stress analyzer. Reduction in total ATP production was also measured. This reveals how important TUM1 is for H2S biosynthesis in the mitochondria of HEK 293T.
Finally, the inhibition of NFS1 in HEK 293T and purified NFS1 protein by 2-methylene 3-quinuclidinone was demonstrated via spectrophotometric and radioactivity quantification. Inhibition of NFS1 by MQ further affected the iron-sulfur cluster-dependent enzyme aconitase activity.