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The increasing loss of pollinators over the last decades has become more and more evident. Intensive use of plant protection products is one key factor contributing to this decline. Especially the mixture of different plant protection products can pose an increased risk for pollinators as synergistic effects may occur. In this study we investigated the effect of the fungicide Cantus® Gold (boscalid/dimoxystrobin), the neonicotinoid insecticide Mospilan® (acetamiprid) and their mixture on honeybees. Since both plant protection products are frequently applied sequentially to the same plants (e.g. oilseed rape), their combination is a realistic scenario for honeybees. We investigated the mortality, the sucrose responsiveness and the differential olfactory learning performance of honeybees under controlled conditions in the laboratory to reduce environmental noise. Intact sucrose responsiveness and learning performance are of pivotal importance for the survival of individual honeybees as well as for the functioning of the entire colony. Treatment with two sublethal and field relevant concentrations of each plant protection product did not lead to any significant effects on these behaviors but affected the mortality rate. However, our study cannot exclude possible negative sublethal effects of these substances in higher concentrations. In addition, the honeybee seems to be quite robust when it comes to effects of plant protection products, while wild bees might be more sensitive.
Highlights
• Mix of SBI fungicides and neonicotinoids can lead to synergistic effects for bees.
• Combination of non-SBI fungicide and neonicotinoid in field-realistic doses tested.
• Synergistic effect on mortality of honeybees.
• No effects on sucrose responsiveness and learning performance of honeybees.
• Synergistic effects by other pesticide mixtures or on wild bees cannot be excluded.
Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.
Introduction
Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear.
Methods
We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved.
Results
Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients.
Discussion
Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.
The narrow escape theory (NET) predicts the escape time distribution of Brownian particles confined to a domain with reflecting borders except for one small window. Applications include molecular activation events in cell biology and biophysics. Specifically, the mean first passage time τ can be analytically calculated from the size of the domain, the escape window, and the diffusion coefficient of the particles. In this study, we systematically tested the NET in a disc by variation of the escape opening. Our model system consisted of micro-patterned lipid bilayers. For the measurement of τ, we imaged diffusing fluorescently-labeled lipids using single-molecule fluorescence microscopy. We overcame the lifetime limitation of fluorescent probes by re-scaling the measured time with the fraction of escaped particles. Experiments were complemented by matching stochastic numerical simulations. To conclude, we confirmed the NET prediction in vitro and in silico for the disc geometry in the limit of small escape openings, and we provide a straightforward solution to determine τ from incomplete experimental traces.
The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.
Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.
Highlights
• Dopamine receptor-1 activation induces TrkB cell-surface expression in striatal neurons
• Dopaminergic deficits cause TrkB accumulation and clustering in the ER
• TrkB clusters colocalize with cargo receptor SORCS-2 in direct pathway striatal neurons
• Intracellular TrkB clusters fail to fuse with lysosomes after dopamine depletion
Summary
Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.
Context
Habitat loss and degradation impose serious threats on biodiversity. However, not all habitats receive the attention commensurate with their ecological importance. Shrub ecotones (successional stages between grasslands and forests) can be highly species-diverse but are often restricted to small areas as prevalent management practices either promote open grassland or forest habitats, threatening the effective conservation of ecotone species.
Objectives
In this study, we assessed the importance of habitat and landscape features of shrub ecotones for the rarely studied true bugs (Heteroptera), a functionally diverse taxon that comprises highly specialized species and broad generalists.
Methods
True bugs were sampled with a beating tray in 118 spatially independent shrub ecotones in a region of 45,000 square kilometers in Germany. In addition to habitat area and landscape context, we used a hedge index to evaluate habitat quality.
Results
Shrub ecotones in open habitats harbored a greater species richness and abundance compared to shaded ones in later seral stages, and species composition differed. Richness and abundance were positively affected by increasing habitat area and quality, whereas an increase in the proportion of semi-natural habitats within 1 km only enhanced richness. While feeding and habitat specialists were more sensitive to habitat area reduction than generalists, this was not the case for weak dispersers and carnivores.
Conclusions
Our findings emphasize the importance of large and high-quality ecotones that form a patchy mosaic of shrubs and herbaceous plants. Such ecotones can benefit both grassland species and species depending on woody plants. Conservation authorities should balance between promoting shrubs and keeping such habitats open to maximize species diversity.
The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood.
Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 % conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts.
In this study, it was demonstrated that the 100 % conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 % conservation of this RNA motif.
A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic.
The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment.
De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis.
SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis.
SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation.
It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth.
A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation.
In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.
Highlights
• The GLA variant S126G is not associated with Fabry symptoms in the presented case
• S126G has no effect on α-GAL A activity or Gb3 levels in this patient
• S126G sensory neurons show no electrophysiological abnormalities
Abstract
Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.
Die vier Crz-Neurone des ventralen Nervensystems von Drosophila melanogaster sammeln Evidenz, wann im Rahmen eines Paarungsakts zirka 6 Minuten vergangen sind. Diese Entscheidung ist für die männliche Fliege von Bedeutung, da das Männchen vor Ablauf dieser ~6 Minuten, welche den Zeitpunkt der Ejakulation darstellen, eher das eigene Leben opfern würde, als dass es die Paarung beenden würde. Nach Ablauf der ~6 Minuten fällt die Motivation des Männchens dagegen dramatisch ab. Im Rahmen der vorliegenden Arbeit wurde zunächst mittels optogenetischer neuronaler Inhibitionsprotokolle sowie Verhaltensanalysen das Phänomen der Evidenz-akkumulation in den Crz-Neuronen genauer charakterisiert. Dabei zeigte sich, dass die akkumulierte Evidenz auch während einer elektrischen Inhibition der Crz-Neurone persistierte. Dieses Ergebnis warf die Hypothese auf, dass das Äquivalent der akkumulierten Evidenz in den Crz-Neuronen biochemischer Natur sein könnte. Es wurde daraufhin ein Hochdurchsatzscreening-Verfahren entwickelt, mittels dessen 1388 genetische Manipulationen der Crz-Neurone durchgeführt und auf eine Änderung der Evidenzakkumulation getestet wurden. Nur ~30 genetische Manipulationen zeigten eine veränderte Evidenzakkumulation, wobei die meisten dieser Manipulationen den cAMP-Signalweg betrafen. Mittels der optogenetischen Photoadenylatzyklase bPAC, einer Reihe weiterer genetischer Manipulationen des cAMP-Signalwegs sowie der ex vivo Kalzium-Bildgebung und Fluoreszenzlebensdauer-Mikroskopie konnte bestätigt werden, dass cAMP das Äquivalent der in den Crz-Neuronen spannungsabhängig akkumulierten Evidenz darstellt, wobei die Kombination dieser Methoden nahelegte, dass der Schwellenwert der Evidenzakkumulation durch die cAMP-Bindungsaffinität der regulatorischen PKA-Untereinheiten festgelegt sein könnte. Mittels genetischer Mosaikexperimente sowie bildgebenden Verfahren konnte darüber hinaus gezeigt werden, dass innerhalb des Crz-Netzwerks eine positive Rückkopplungsschleife aus rekurrenter Aktivität sowie der cAMP-Akkumulation besteht, welche, sobald die cAMP-Spiegel den Schwellenwert erreichen, zu einem netzwerkweit synchronisierten massiven Kalziumeinstrom führt, was die Abgabe des Crz-Signals an nachgeschaltete Netzwerke triggert. Dieses Phänomen könnte ein Analogon des Aktionspotenzials auf Netzwerkebene sowie auf Intervallzeitskalen darstellen und wurde als „Eruption“ bezeichnet. Genetische, optogenetische sowie Bildgebungsexperimente konnten zeigen, dass die CaMKII derartige Eruptionen durch Niedrighalten der cAMP-Spiegel unterdrückt, was den Zeitmessmechanismus des ersten beschriebenen Intervallzeitmessers CaMKII offenlegt.
Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins.Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an “all in one” solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody-accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal a history of dynamic protein interaction.In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment.
The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.
A novel USP11-TCEAL1-mediated mechanism protects transcriptional elongation by RNA Polymerase II
(2024)
Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge.
Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding.
In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS.
Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma.
Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\)
(2024)
The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface.
In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information.
In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded.
I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found.
Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton.
The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed.
Proteins fold in water and achieve a clear structure despite a huge parameter space. Inside a (protein) crystal you have everywhere the same symmetries as there is everywhere the same unit cell. We apply this to qubit interactions to do fundamental physics:
We modify cosmological inflation: we replace the big bang by a condensation event in an eternal all-encompassing ocean of free qubits. Rare interactions of qubits in the ocean provide a nucleus or seed for a new universe (domain), as the qubits become decoherent and freeze-out into defined bit ensembles. Next, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth). The crystal unit cell guarantees same symmetries (and laws of nature) everywhere inside the crystal, no inflation scenario is needed.
Interacting qubits solidify, quantum entropy decreases in the crystal, but increases outside in the ocean. The interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After this very early modified steps, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements.
Applying the Hurwitz theorem to qubits we prove that initiation of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. We describe a six-bit-ensemble toy model of qubit interaction and the repulsive forces of qubits for ultra-short distances. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below Planck´s quantum is liquidity left). The E8 symmetry of heterotic string theory has six curled-up, small dimensions. These keep the qubit crystal together and never expand. We give energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit crystal formation.
Implications are fundamental answers, e.g. why there is fine-tuning for life-friendliness, why there is string theory with rolled-up dimension and so many free parameters. We explain by cosmological crystallization instead of inflation the early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: the unit cell of our crystal universe has a matter handedness avoiding anti-matter. Importantly, crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness. Vacuum energy gets appropriate low inside the crystal by its qubit binding energy, outside it is 10**20 higher. Scalar fields for color interaction/confinement and gravity could be derived from the qubit-interaction field.
The Western Honeybee (Apis mellifera) is among the most versatile species in the world. Its adaptability is rooted in thousands of the differently specialized individuals acting jointly together. Thus, bees that are able to handle a certain task or condition well can back up other individuals less capable to do so on the colony level. Vice versa, the latter individuals might perform better in other situations. This evolutionary recipe for success ensures the survival of colonies despite challenging habitat conditions. In this context, the ectoparasitic mite Varroa destructor reflects the most pronounced biotic challenge to honeybees worldwide. Without proper treatment, infested colonies rapidly dwindle and ultimately die. Nevertheless, resistance behaviours against this parasite have evolved in some populations through natural selection, enabling colonies to survive untreated. In this, different behaviours appear to be adapted to the respective habitat conditions and may complement each other. Yet, the why and how of this behavioural response to the mite remains largely unknown. My thesis focuses on the biological background of Varroa-resistance traits in honeybees and presents important findings for the comprehension of this complex host-parasite interaction. Based on this, I draw implications for both, applied bee breeding and scientific investigations in the field of Varroa-resistance. Specifically, I focus on two traits commonly found in resistant and, to a lower degree, also mite-susceptible colonies: decreased mite reproduction and the uncapping and subsequent recapping of sealed brood cells. Examining failures in the reproductive success of mites as a primary mechanism of Varroa-resistance, I was able to link them to specific bee behaviours and external factors. Since mite reproduction and the brood rearing of bees are inevitably connected, I first investigated the effects of brood interruption on the reproductive success of mites. Brood interruption decreased the reproductive success of mites both immediately and in the long term. By examining the causes of reproductive failure, I could show that this was mainly due to an increased share of infertile mites. Furthermore, I proved that interruption in brood rearing significantly increased the expression of recapping behaviour. These findings consequently showed a dynamic modulation of mite reproduction and recapping, as well as a direct effect of brood interruption on both traits. To further elucidate the plasticity in the expression of both traits, I studied mite reproduction, recapping behaviour and infestation levels over the course of three years. The resulting extensive dataset unveiled a significant seasonal variation in mite reproduction and recapping. In addition, I show that recapping decreases the reproductive success of mites by increasing delayed developing female offspring and cells lacking male offspring. By establishing a novel picture-based brood investigation method, I could furthermore show that both the removal of brood cells and recapping activity specifically target brood ages in which mite offspring would be expected. Recapping, however, did not cause infertility of mites. Considering the findings of my first study, this points towards complementary mechanisms.
This underlines the importance of increased recapping behaviour and decreased mite reproduction as resistance traits, while at the same time emphasising the challenges of reliable data acquisition. To pave the way for a practical application of these findings in breeding, we then investigated the heritability (i.e., the share of genotypic variation on the observed phenotypic variation) of the accounted traits. By elaborating comparable test protocols and compiling data from over 4,000 colonies, we could, for the first time, demonstrate that recapping of infested cells and decreased reproductive success of mites are heritable (and thus selectable) traits in managed honeybee populations.
My thesis proves the importance of recapping and decreased mite reproduction as resistance traits and therefore valuable goals for breeding efforts. In this regard, I shed light on the underlying mechanisms of both traits, and present clear evidence for their interaction and heritability.
The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner.
In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes.
Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance.
Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth.
To grow larger, insects must shed their old rigid exoskeleton and replace it with a new one. This process is called molting and the motor behavior that sheds the old cuticle is called ecdysis. Holometabolic insects have pupal stages in between their larval and adult forms, during which they perform metamorphosis. The pupal stage ends with eclosion, i.e., the emergence of the adult from the pupal shell. Insects typically eclose at a specific time during the day, likely when abiotic conditions are at their optimum. A newly eclosed insect is fragile and needs time to harden its exoskeleton. Hence, eclosion is regulated by sophisticated developmental and circadian timing mechanisms.
In Drosophila melanogaster, eclosion is limited to a daily time window in the morning, regarded as the “eclosion gate”. In a population of laboratory flies entrained by light/dark cycles, most of the flies eclose around lights on. This rhythmic eclosion pattern is controlled by the circadian clock and persists even under constant conditions.
Developmental timing is under the control of complex hormonal signaling, including the steroid ecdysone, insulin-like peptides, and prothoracicotropic hormone (PTTH). The interactions of the central circadian clock in the brain and a peripheral clock in the prothoracic gland (PG) that produces ecdysone are important for the circadian timing of eclosion. These two clocks are connected by a bilateral pair of peptidergic PTTH neurons (PTTHn) that project to the PG. Before each molt, the ecdysone level rises and then falls shortly before ecdysis. The falling ecdysone level must fall below a certain threshold value for the eclosion gate to open. The activity of PTTHn is inhibited by short neuropeptide F (sNPF) from the small ventrolateral neurons (sLNvs) and inhibition is thought to lead to a decrease in ecdysone production.
The general aim of this thesis is to further the understanding of how the circadian clock and neuroendocrinal pathways are coordinated to drive eclosion rhythmicity and to identify when these endocrinal signaling pathways are active. In Chapter I, a series of conditional PTTHn silencing-based behavioral assays, combined with neuronal activity imaging techniques such as non-invasive ARG-Luc show that PTTH signaling is active and required shortly before eclosion and may serve to phase-adjust the activity of the PG at the end of pupal development. Trans-synaptic anatomical stainings identified the sLNvs, dorsal neurons 1 (DN1), dorsal neurons 2 (DN2), and lateral posterior neurons (LPNs) clock neurons as directly upstream of the PTTHn.
Eclosion motor behavior is initiated by Ecdysis triggering hormone (ETH) which activates a pair of ventromedial (Vm) neurons to release eclosion hormone (EH) which positively feeds back to the source of ETH, the endocrine Inka cells. In Chapter II trans-synaptic tracing showed that most clock neurons provide input to the Vm and non-canonical EH neurons. Hence, clock can potentially influence the ETH/EH feedback loop. The activity profile of the Inka cells and Vm neurons before eclosion is described. Vm and Inka cells are active around seven hours before eclosion. Interestingly, all EH neurons appear to be exclusively peptidergic.
In Chapter III, using chemoconnectomics, PTTHns were found to express receptors for sNPF, allatostatin A (AstA), allatostatin C (AstC), and myosuppressin (Ms), while EH neurons expressed only Ms and AstA receptors. Eclosion assays of flies with impaired AstA, AstC, or Ms signaling do not show arrhythmicity under constant conditions. However, optogenetic activation of the AstA neurons strongly suppresses eclosion.
Chapter IV focuses on peripheral ventral’ Tracheal dendrite (v’Td) and class IV dendritic arborization (C4da) neurons. The C4da neurons mediate larval light avoidance through endocrine PTTH signaling. The v’Td neurons mainly receive O2/CO2 input from the trachea and are upstream of Vm neurons but are not required for eclosion rhythmicity. Conditional ablation of the C4da neurons or torso (receptor of PTTH) knock-out in the C4da neurons impaired eclosion rhythmicity. Six to seven hours before eclosion, PTTHn, C4da, and Vm neurons are active based on ARG-Luc imaging. Thus, C4da neurons may indirectly connect the PTTHn to the Vm neurons.
In summary, this thesis advances our knowledge of the temporal activity and role of PTTH signaling during pupal development and rhythmic eclosion. It further provides a comprehensive characterization of the synaptic and peptidergic inputs from clock neurons to PTTHn and EH neurons. AstA, AstC, and Ms are identified as potential modulators of eclosion circuits and suggest an indirect effect of PTTH signaling on EH signaling via the peripheral sensory C4da neurons.