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- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
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- Research Center for Infectious Diseases, University of Wuerzburg, Wuerzburg 97080, Germany (1)
- Rudolf-Virchow-Zentrum für Experimentelle Biomedizin der Universität Würzburg (1)
Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona Löffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic
stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second
PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes
overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases.
Background
Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent.
Methods
Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices.
Results
The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin.
Conclusions
Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis. "
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.
The thesis provides insights in reconstruction and analysis pipelines for processing of
three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone.
The images were captured in a Light Sheet Fluorescence Microscope. The work
presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of
the University of Würzburg, performed at the Rudolf-Virchow Center. Despite ongoing
research within the field of megakaryopoiesis, its spatio-temporal pattern of
megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to
promote development of new therapeutic strategies for conditions related to
thrombocytopathy as well as thrombocytopenia. The current concept of
megakaryopoiesis is largely based on data from cryosectioning or in vitro studies
indicating the existence of spatial niches within the bone marrow where specific stages
of megakaryopoiesis take place. Since classic imaging of bone sections is typically
limited to selective two-dimensional views and prone to cutting artefacts, imaging of
intact murine bone is highly desired. However, this has its own challenges to meet,
particularly in image reconstruction. Here, I worked on processing pipelines to account
for irregular specimen staining or attenuation as well as the extreme heterogeneity of
megakaryocyte morphology. Specific challenges for imaging and image reconstruction
are tackled and solution strategies as well as remaining limitations are presented and
discussed. Fortunately, modern image processing and segmentation strongly benefits
from continuous advances in hardware as well as software-development. This thesis
exemplifies how a combined effort in biomedicine, computer vision, data processing
and image technology leads to deeper understanding of megakaryopoiesis. Tailored
imaging pipelines significantly helped elucidating that the large megakaryocytes are
broadly distributed throughout the bone marrow facing a surprisingly dense vessel
network. No evidence was found for spatial niches in the bone marrow, eventually
resulting in a revised model of megakaryopoiesis.
Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.
Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity
(2018)
Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others, activates G-protein coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the β3-adrenergic receptor (ADRB3) in a CCAAT/enhancerbinding protein (C/EBP)-α and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, loss of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.
Background:
Inhibition of early platelet adhesion by blockade of glycoprotein-IB (GPIb) protects mice from ischemic stroke. To elucidate underlying mechanisms in-vivo, infarct development was followed by ultra-high field MRI at 17.6 Tesla.
Methods:
Cerebral infarction was induced by transient-middle-cerebral-artery-occlusion (tMCAO) for 1 hour in C57/BL6 control mice (N = 10) and mice treated with 100 mg Fab-fragments of the GPIb blocking antibody p0p/B 1 h after tMCAO (N = 10). To control for the effect of reperfusion, additional mice underwent permanent occlusion and received anti-GPIb treatment (N = 6; pMCAO) or remained without treatment (N = 3; pMCAO). MRI 2 h and 24 h after MCAO measured cerebral-blood-flow (CBF) by continuous arterial-spin labelling, the apparent-diffusion-coefficient (ADC), quantitative-T2 and T2-weighted imaging. All images were registered to a standard mouse brain MRI atlas and statistically analysed voxel-wise, and by cortico-subcortical ROI analysis.
Results:
Anti-GPIb treatment led to a relative increase of postischemic CBF vs. controls in the cortical territory of the MCA (2 h: 44.2 +/- 6.9 ml/100g/min versus 24 h: 60.5 +/- 8.4; p = 0.0012, F((1,18)) = 14.63) after tMCAO. Subcortical CBF 2 h after tMCAO was higher in anti-GPIb treated animals (45.3 +/- 5.9 vs. controls: 33.6 +/- 4.3; p = 0.04). In both regions, CBF findings were clearly related to a lower probability of infarction (Cortex/Subcortex of treated group: 35%/65% vs. controls: 95%/100%) and improved quantitative-T2 and ADC. After pMCAO, anti-GPIb treated mice developed similar infarcts preceded by severe irreversible hypoperfusion as controls after tMCAO indicating dependency of stroke protection on reperfusion.
Conclusion:
Blockade of platelet adhesion by anti-GPIb-Fab-fragments results in substantially improved CBF early during reperfusion. This finding was in exact spatial correspondence with the prevention of cerebral infarction and indicates in-vivo an increased patency of the microcirculation. Thus, progression of infarction during early ischemia and reperfusion can be mitigated by anti-platelet treatment.
Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2\(^{+}\)-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation". This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.
Background:
We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, beta-galactosidase, and beta-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV 1h68. This strain shows tumor specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice
Methods:
A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV 1h68 with short non coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.
Results:
we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.
Conclusions:
These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.
Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane.
Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand.
Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.
Objectives. In diabetes accumulated advanced glycation end products (AGEs) are involved in the striking cardiovascular morbidity/mortality. We asked whether a hypovitaminosis D associates with an increased formation and toxicity of AGEs in diabetes.
Methods. In 276 diabetics (160M/116 F, age: 65.0 ± 13.4; 43 type 1,T1DM, and 233 type 2 patients, T2DM) and 121 nondiabetic controls (60 M/61 F; age: 58.6 ± 15.5 years) routine biochemistry, levels of 25-hydroxyvitamin D\(_{3}\) (25-(OH)D), skin autofluorescence (SAF), plasma AGE-associated fluorescence (AGE-FL), N\(^{ε}\) -(carboxymethyl) lysine (CML), soluble receptor for AGEs (sRAGE), soluble vascular adhesion protein-1 (sVAP-1), high sensitive C-reactive protein (hs-CRP), and renal function (eGFR) were determined.
Results. In the diabetics SAF and AGE-Fl were higher than those of the controls and correlated with age, duration of diabetes, and degree of renal impairment. In T2DM patients but not in T1DM the age-dependent rise of SAF directly correlated with hs-CRP and sVAP-1. 25-(OH)D levels in diabetics and nondiabetics were lowered to a similar degree averaging 22.5 ng/mL. No relationship between 25-(OH)D and studied markers except for sVAP-1 was observed in the diabetics. Conclusion. In diabetics hypovitaminosis D does not augment accumulation of AGEs and studied markers of microinflammation and oxidative stress except for sVAP-1.
The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use singlemolecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.
Background:
Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties.
Methods and Findings:
Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors.
Conclusions:
Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.
Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.
Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4\(^+\) or CD8\(^+\) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b\(^+\) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b\(^+\) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.
Visual information is essential for Drosophila to navigate its environment. The visual system of the fly has been studied for many decades and has yielded many insights about vision in general. However, visual information can be ambiguous and the system processing it needs to be able to cope with that. In this study, the visual orientation behavior of Drosophila is challenged by panoramic incoherent motion stimuli to which the fly can respond in three different, equally adaptive ways. The study is conducted in a well-established setup, the so-called flight simulator (Heisenberg and Wolf, 1993), where the fly can control its visual surroundings in stationary flight with its yaw torque, which is simultaneously recorded. The fly can either use one of two incoherently moving panorama patterns or the integrated motion of both as its reference for straight flight. It is observed that flies use all three of these behavioral alternatives for orientation. Previous models of fly motion vision do not predict a bimodal tuning to incoherent wide-field motion stimuli (Joesch et al., 2008, Borst et al., 1995), however, a recent study on blowflies could suggests that they show component selectivity to the individual moving gratings in a compound plaid stimulus (Saleem et al., 2012). Here, it can be shown that the same bimodal tuning manifests in Drosophila, although the stimuli used are different and most of the experiments are conducted in closed loop. It is found that the extent to which the Drosophila expresses this component selectivity in its orientation behavior, i.e. how often it stabilizes a single panorama pattern instead of the integrated motion of both, depends on two properties of the panorama stimuli, pattern contrast and horizontal pattern element distance. Single pattern stabilization decreases with increasing contrast and increasing pattern element distance. In the latter case, it increases again when there are very few horizontal pattern elements, although that appears to be the result of a lack of rivalry between the patterns due to the low number of pattern elements. Both increased pattern contrast and pattern element distance increase the salience of the single pattern elements. A single element in a compound visual stimulus, like a dot within a dot pattern, can be interpreted as a standalone figure or a part of a bigger unit. Previous studies on Drosophila vision have concentrated on how the fly discriminates a figure from the background (Heisenberg and Wolf, 1984, Bahl et al., 2013, Aptekar et al., 2012), but have hardly touched the question of what qualifies a figure or a background (i.e. a panorama) stimulus as such. In the present study, it is observed that, when exposed to incoherent panoramic motion stimuli, the flies prefer to orient themselves towards the average of the two motions when the panorama stimuli possess strong figure features and towards the single patterns when they do not and single pattern elements are therefore less salient.
The above-mentioned plaid stimuli are a well-known multistable percept in human psychophysics. Multistability is a property of higher visual systems and considered an indicator of endogenous activity in vision. As Drosophila expresses behavioral multistability in the IPMP, it is evaluated in this respect. The results show several parallels to human multistable perception. For one, the frequency and duration with which a behavior occurs, can be influenced, but the occurrence of the behaviors is non-deterministic and not coupled to the stimulus. It can also be shown that the switches between behaviors do not stem from a rivalry of the two visual hemispheres of the fly, although monocularity does also influence the likelihood with which the behaviors occur. Secondly, like in human perceptual rivalry, individual flies exhibit strong idiosyncrasies regarding the overall durations they spend with the different behaviors and the frequencies with which they switch between them. Finally, the distribution of the durations between the behavioral switches can be fit to the same function as the distribution of percept durations in human multistable perception, the gamma function, although it has a different shape and therefore also differing parameters. The Drosophila mutant radish, which has been shown to have attention-like deficits (van Swinderen and Brembs, 2010, Koenig et al., 2016a), does also express an altered behavior in the IPMP compared to wildtype flies. As these behavioral alterations resemble effects on multistable perception found in humans suffering from ADHD (Amador-Campos et al., 2015) and perceptual multistability is generally considered to be closely related to attention (Leopold and Logothetis, 1999), attentional processes are also very likely to play a role in the flies’ behavior in the IPMP.
In conclusion, the visual system of Drosophila is capable disentangle incoherent motion stimuli even if they overlap and cover the entire visual field, i.e. it shows component selectivity of wide-field motion. Whether it uses a single wide-field motion component or the average of two as its reference for straight flight depends on pattern contrast and horizontal pattern element density, which indicates an involvement of a figure-background rivalry. This rivalry and the one between the two wide-field motion components elicit a multistability in the orientation behavior of the fly the temporal dynamics of which partially resemble the temporal dynamics of human multistable perception and which also suggests the involvement of attentional processes.
Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.
Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.
Platelet aggregation at sites of vascular injury is essential to limit posttraumatic blood loss, but may also cause acute ischemic disease states such as myocardial infarction or stroke. Stable thrombus formation requires a series of molecular events involving platelet receptors and intracellular signal transduction, which contribute to adhesion, activation and aggregation of platelets. In this thesis, the cellular regulation of platelet surface receptors and their involvement in thrombus formation was investigated using genetically modified mice.
In the first part of the study, the functional relevance of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and of the recently identified hemITAM-bearing C-type lectin-like receptor 2 (CLEC-2) for in vivo thrombus formation was analyzed. Megakaryocyte/ platelet-specific CLEC-2 knock out mice displayed a defective lymphatic development and were protected from occlusive arterial thrombus formation. These phenotypes were more pronounced in mice with a GPVI/CLEC-2 double deficiency. Hemostasis was not compromised in CLEC-2 or GPVI single-deficient animals, as they showed only mildly prolonged tail bleeding times. Combined depletion of both receptors resulted in markedly prolonged bleeding times revealing an unexpected redundant function of the two receptors in hemostasis as well as thrombosis. These findings might have important implications for the development of anti-CLEC-2/ anti-GPVI agents as therapeutics.
In the second part, mechanisms underlying the cellular regulation of CLEC-2 were studied. Previous studies have shown that injection of the anti-CLEC-2 antibody INU1 results in complete immunodepletion of platelet CLEC-2 in mice, which is preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. It is demonstrated that INU1-induced CLEC-2 immunodepletion occurs through Src family kinase (SFK)-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with spleen tyrosine kinase (Syk) deficiency, INU1-induced CLEC-2 internalization/ degradation was fully preserved, whereas the associated thrombocytopenia was largely prevented. These results show that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia.
Since INU1 IgG induced a pronounced thrombocytopenia, the in vivo effects of monovalent INU1 F(ab) fragments were analyzed. Very unexpectedly, injection of the F(ab) fragments resulted in widespread thrombus formation leading to persistent neurological deficits of the animals. This intravascular thrombus formation is the result of CLEC-2-dependent platelet activation and aggregation. The mechanism underlying the thrombus formation is still unknown and depends potentially on binding of a yet unidentified ligand to F(ab)-opsonized CLEC-2 on platelets.