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Sonstige beteiligte Institutionen
Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis.
First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level.
Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice.
In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation.
Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.
Functional analysis of polarization and podosome formation of murine and human megakaryocytes
(2019)
In mammals, blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MK) that extend polarized cell protrusions (proplateles) into BM sinusoids. Proplatelet formation (PPF) requires substantial cytoskeletal rearrangements that have been shown to involve the formation of podosomes, filamentous actin (F-actin) and integrin-rich structures. However, the exact molecular mechanisms regulating MK podosome formation, polarization and migration within the BM are poorly defined. According to current knowledge obtained from studies with other cell types, these processes are regulated by Rho GTPase proteins like RhoA and Cdc42.
In this thesis, polarization and podosome formation were investigated in MKs from genetically modified mice, as well as the cell lines K562 and Meg01 by pharmacological modulation of signaling pathways.
The first part of this thesis describes establishment of the basic assays for investigation of MK polarization. Initial data on polarization of the MK-like erythroleukemia cell line K562 revealed first insights into actin and tubulin dynamics of wild type (WT) and RhoA knock-out (RhoA-/-) K562 cells. Phorbol 12-myristate 13-acetate (PMA)-induction of K562 cells led to the expected MK-receptor upregulation but also RhoA depletion and altered polarization patterns.
The second part of this thesis focuses on podosome formation of MKs. RhoA is shown to be dispensable for podosome formation. Cdc42 is revealed as an important, but not essential regulator of MK spreading and podosome formation. Studies of signaling pathways of podosome formation reveal the importance of the tyrosine kinases Src, Syk, as well as glycoprotein (GP)VI in MK spreading and podosome formation.
This thesis provides novel insights into the mechanisms underlying polarization and podosome formation of MKs and reveals new, important information about cytoskeletal dynamics of MKs and potentially also platelets.
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.
The aim of this thesis was the preparation of a biomaterial ink for the fabrication of chemically crosslinked hydrogel scaffolds with low micron sized features using melt electrowriting (MEW). By developing a functional polymeric material based on 2-alkyl-2-oxazine (Ozi) and 2-alkyl-2-oxazoline (Ox) homo- and copolymers in combination with Diels-Alder (DA)-based dynamic covalent chemistry, it was possible to achieve this goal. This marks an important step for the additive manufacturing technique melt electrowriting (MEW), as soft and hydrophilic structures become available for the first time. The use of dynamic covalent chemistry is a very elegant and efficient method for consolidating covalent crosslinking with melt processing. It was shown that the high chemical versatility of the Ox and Ozi chemistry offers great potential to control the processing parameters. The established platform offers straight forward potential for modification with biological cues and fluorescent markers. This is essential for advanced biological applications. The physical properties of the material are readily controlled and the potential for 4D-printing was highlighted as well. The developed hydrogel architectures are excellent candidates for 3D cell culture applications. In particular, the low internal strength of some of the scaffolds in combination with the tendency of such constructs to collapse into thin strings could be interesting for the cultivation of muscle or nerve cells. In this context it was also possible to show that MEW printed hydrogel scaffolds can withstand the aspiration and ejection through a cannula. This allows the application as scaffolds for the minimally invasive delivery of implants or functional tissue equivalent structures to various locations in the human body.
Schicksal von Mikrokernen bzw. mikrokernhaltigen Zellen und Bedeutung von Mikrokernen als Biomarker
(2021)
Mikrokerne sind als wichtiger Biomarker in der Gentoxizitätsforschung seit langer Zeit etabliert und ihre Bildung ist mechanistisch gut verstanden, wohingegen das Mikrokernschicksal und die genaue Funktion von Mikrokernen in der Kanzerogenese unzureichend erforscht sind. Um das Schicksal von Mikrokernen und mikrokernhaltigen Zellen über einen längeren Zeitraum zu untersuchen, wurden HeLa-Zellen, die mit einem GFP-markierten Histon H2B transfiziert worden sind, mittels Lebendzellmikroskopie nach Behandlung mit verschiedenen gentoxischen Agenzien für 96 h untersucht. Parameter wie die Mitose- oder Zelltodrate wurden dabei ebenso wie das Schicksal der Mikrokerne dokumentiert. Während Persistenz und Reinkorporation von Mikrokernen häufig beobachtet wurden, waren Degradation und Auswurf von Mikrokernen selten bis gar nicht zu sehen. Auch konnte ein Teil der mikrokernhaltigen Zellen über mehrere Zellteilungen persistieren und proliferieren, wodurch die in Mikrokernen manifestierte chromosomale Instabilität unverändert bleiben kann. Ein eindeutiger Substanzeinfluss auf das Mikrokernschicksal konnte nicht ausgemacht werden. Extrusion sollte weiterhin durch Behandlung mit Hydroxyurea oder Cytochalasin B in Kombination mit gentoxischer Behandlung induziert werden, es wurde jedoch kein Effekt auf die Extrusionsrate beobachtet. Degradation wurde mittels γH2AX-Antikörperfärbung und transduziertem dsRed-markierten Autophagiemarker LC3B in HeLa-H2B-GFP-Zellen untersucht. Trotz erhöhter DNA-Degradation in Mikrokernen wurde nur selten eine Ko-Lokalisierung mit LC3B beobachtet. Dafür gab es in HeLa-H2B-GFP-Zellen, die zusätzlich mit dsRed markierten Kernmembranmarker Lamin B1 transduziert worden sind, Anzeichen für eine eingeschränkte Mikrokernmembranintegrität. Weiterhin wurden Zytokinese-Block Mikrokerntests nach Behandlung mit Thebain mit und ohne metabolische Aktivierung sowie Celecoxib und Celecoxibderivaten durchgeführt. Hierbei wurde nach Thebainbehandlung nur ohne metabolische Aktivierung und bei Anwesenheit von Zytotoxizität mehr Mikrokerne gefunden, während nach Behandlung mit Celecoxib und Celecoxibderivaten kein Anstieg beobachtet wurde. Zusätzlich wurde der Einfluss durch neurodegenerative Veränderungen auf Mundschleimhautzellen in zwei großen Kohorten untersucht, wobei keine Effekte auf die Häufigkeit von Mikrokernen oder mikrokernhaltigen Zellen zugeordnet werden konnten, während es teilweise bei Parametern, die auf Zytotoxizität hindeuten, zu Veränderungen kam. Es konnte insgesamt gezeigt werden, dass Mikrokerne und mikrokernhaltige Zellen zusätzlich zu ihrer Funktion als Biomarker über wenigstens mehrere Zellteilungen bestehen bleiben können. Auf diese Weise können sie z. B. über Chromothripsis zu einer beschleunigten Kanzerogenese führen, was zu einer schlechten Prognose für Krebspatienten führen kann.
The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches.
Platelets are the second most abundant blood cells and their main function is maintenance of vascular integrity. In addition, platelets are increasingly recognized as cells with immune functions, as they participate in the recruitment of immune cells and modulate the progression and severity of an immune response. So-called lipid mediators, which are – besides other cells – released by activated platelets, influence the immune response. LTB4 is one of these potent lipid mediators and is able to activate neutrophils and induce their infiltration into injured tissue.
In order to investigate the involvement of platelets in inflammatory processes, a murine model of hepatic ischemia reperfusion injury as well as confocal intravital microscopy of the liver were established. Both methods were used to analyze the influence of platelets on the inflammation that follows sterile liver inflammation. We found platelet function to be unaltered after three hours of reperfusion and platelet aggregation to be irrelevant for the outcome of hepatic ischemia reperfusion injury. However, a strong impact of the GPIb-vWF axis could be observed, as antibody mediated blockade of GPIb as well as vWF-deficiency significantly reduced liver damage markers and decreased neutrophil infiltration. GPIb-IL-4R mice were used to exclude the possibility that the protective effects of the anti-GPIbα antibody treatment (p0p/B) results from something else than blocking GPIbα. Furthermore, the slope of neutrophil infiltration was decreased in p0p/B-treated mice, leading to overall decreased neutrophil numbers in the liver after three hours of reperfusion. Blockade of the integrin αIIbβ3, however, showed no reduction in neutrophil infiltration into the post-ischemic liver, in line with unaltered liver damage.
To study the role of leukotriene B4, conditional and constitutive knockout mice for the LTA4 hydrolase, which catalyzes the last step in LTB4 synthesis, were generated. Lta4h deficiency did not affect general platelet functionality in hemostasis and thrombosis. Interestingly,
Lta4h-/- mice were not protected from cellular damage following hepatic ischemia, despite lower neutrophil numbers in the post-ischemic liver.
Intravital microscopy of the pancreas was established and revealed increased CD4+ T cell numbers in GPVI-deficient animals compared to WT controls in line with the pre-diabetic phenotype of Gp6-/- mice that was revealed in Grzegorz Sumara’s group. Furthermore, platelet ‘behavior’ in pancreatic islets was observed following glucose injection. We found a high number of platelets adherent to islet sinusoids under basal conditions and no rolling/decelerating of platelets following glucose injection. This was accompanied by temporary sinusoidal constriction and stop of the blood flow. This phenomenon was not observed in control settings (injection of PBS, insulin or L-glucose).
In a side project, which was carried out jointly with Tobias Heib, a side by side comparison of the classical syringe-based flushing and the centrifugation-based spinning method to isolate murine bone marrow was conducted. Flow cytometry revealed no differences in the distribution of hematopoietic stem cells and immune cells and functional analysis with primary and cultured megakaryocytes (MKs) showed comparable results in all conducted assays. Thus, our data demonstrated that the faster and more efficient spinning method can be used for the isolation of bone marrow cells.
Stroke and myocardial infarction are the most prominent and severe consequences of pathological thrombus formation. For prevention and/or treatment of thrombotic events there is a variety of anti-coagulation and antiplatelet medication that all have one side effect in common: the increased risk of bleeding. To design drugs that only intervene in the unwanted aggregation process but do not disturb general hemostasis, it is crucial to decipher the exact clotting pathway which has not been fully understood yet. Platelet membrane receptors play a vital role in the clotting pathway and, thus, the aim of this work is to establish a method to elucidate the interactions, clustering, and reorganization of involved membrane receptors such as GPIIb/IIIa and GPIX as part of the GPIb-IX-V complex. The special challenges regarding visualizing membrane receptor interactions on blood platelets are the high abundancy of the first and the small size of the latter (1—3µm of diameter). The resolution limit of conventional fluorescence microscopy and even super-resolution approaches prevents the successful differentiation of densely packed receptors from one another. Here, this issue is approached with the combination of a recently developed technique called Expansion Microscopy (ExM). The image resolution of a conventional fluorescence microscope is enhanced by simply enlarging the sample physically and thus pulling the receptors apart from each other. This method requires a complex sample preparation and holds lots of obstacles such as variable or anisotropic expansion and low images contrast. To increase ExM accuracy and sensitivity for interrogating blood platelets, it needs optimized sample preparation as well as image analysis pipelines which are the main part of this thesis. The colocalization results show that either fourfold or tenfold expanded, resting platelets allow a clear distinction between dependent, clustered, and independent receptor organizations compared to unexpanded platelets.Combining dual-color Expansion and confocal fluorescence microscopy enables to image in the nanometer range identifying GPIIb/IIIa clustering in resting platelets – a pattern that may play a key role in the clotting pathway
Sharpening super-resolution by single molecule localization microscopy in front of a tuned mirror
(2020)
The „Resolution Revolution" in fluorescence microscopy over the last decade has given rise to a variety of techniques that allow imaging beyond the diffraction limit with a resolution power down into the nanometer range. With this, the field of so-called super-resolution microscopy was born. It allows to visualize cellular architecture at a molecular level and thereby achieve a resolution level that had been previously only accessible by electron microscopy approaches.
One of these promising techniques is single molecule localization microscopy (SMLM) in its most varied forms such as direct stochastic optical reconstruction microscopy (dSTORM) which are based on the temporal separation of the emission of individual fluorophores. Localization analysis of the subsequently taken images of single emitters eventually allows to reconstruct an image containing super-resolution information down to typically 20 nm in a cellular setting. The key point here is the localization precision, which mainly depends on the image contrast generated the by the individual fluorophore’s emission. Thus, measures to enhance the signal intensity or reduce the signal background allow to increase the image resolution achieved by dSTORM. In my thesis, this is achieved by simply adding a reflective metal-dielectric nano-coating to the microscopy coverslip that serves as a tunable nano-mirror.
I have demonstrated that such metal-dielectric coatings provide higher photon yield at lower background and thus substantially improve SMLM performance by a significantly increased localization precision, and thus ultimately higher image resolution. The strength of this approach is that ─ except for the coated cover glass ─ no specialized setup is required. The biocompatible metal-dielectric nano-coatings are fabricated directly on microscopy coverslips and have a simple three-ply design permitting straightforward implementation into a conventional fluorescence microscope. The introduced improved lateral resolution with such mirror-enhanced STORM (meSTORM) not only allows to exceed Widefield and Total Internal Reflection Fluorescence (TIRF) dSTORM performance, but also offers the possibility to measure in a simplified setup as it does not require a special TIRF objective lens.
The resolution improvement achieved with meSTORM is both spectrally and spatially tunable and thus allows for dual-color approaches on the one hand, and selectively highlighting region above the cover glass on the other hand, as demonstrated here.
Beyond lateral resolution enhancement, the clear-cut profile of the highlighted region provides additional access to the axial dimension. As shown in my thesis, this allows for example to assess the three-dimensional architecture of the intracellular microtubule network by translating the local localization uncertainty to a relative axial position. Even beyond meSTORM, a wide range of membrane or surface imaging applications may benefit from the selective highlighting and fluorescence enhancing provided by the metal-dielectric nano-coatings. This includes for example, among others, live-cell Fluorescence Correlation Spectroscopy and Fluorescence Resonance Energy Transfer studies as recently demonstrated.
Ranvier-Schnürringe spielen eine entscheidende Rolle bei der schnellen Weiterleitung von elektrischen Impulsen in Nervenzellen. Bei bestimmten neurologischen Erkrankungen, den Neuropathien, kann es zu Störungen in der ultrastrukturellen Organisation verschiedener Schnürring-Proteine kommen (Doppler et al., 2018, Doppler et al., 2016).
Eine detailliertere Kenntnis der genauen Anordnung dieser Schnürring-Proteine und eventueller Abweichungen von dieser Anordnung im Krankheitsfall, könnte der Schlüssel zu einer vereinfachten Diagnostik von bestimmten Neuropathie- Formen sein.
Ziel meiner Arbeit war es daher, die Untersuchung der ultrastrukturellen Architektur der (para-)nodalen Adhäsionsproteine Neurofascin-155 und Caspr1 unter Verwendung der super-hochauflösenden Mikroskopiemethode dSTORM (direct Stochastic Optical Reconstruction Microscopy) an murinen Zupfnervenpräparaten zu etablieren. Nach erster Optimierung der Probenpräparation für die 2-Farben-dSTORM sowie der korrelationsbasierten Bildanalyse, konnte ich mittels modellbasierter Simulation die zugrundeliegende Molekülorganisation identifizieren und mit Hilfe der Ergebnisse aus früheren Untersuchungen validieren. In einem translationalen Ansatz habe ich anschließend humane Zupfnervenpräparate von 14 Probanden mit unterschiedlichen Formen einer Neuropathie mikroskopiert und ausgewertet, um die Anwendbarkeit dieses Ansatzes in der Diagnostik zu testen.
Obgleich keine signifikanten Unterschiede zwischen physiologischem und pathologischem neurologischem Gewebe hinsichtlich Neurofascin-155 und Caspr1 festgestellt werden konnten, scheint der Ansatz grundsätzlich dennoch vielversprechend zu sein, bedarf jedoch noch weiteren Anstrengungen hinsichtlich Probenpräparation, Auswertungs- und Versuchsprotokollen und einer größeren Anzahl an humanen Biopsien mit homogenerem Krankheitsbild.