Biologische Hochschulschriften (Goethe-Universität)
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Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
Gepulste dipolare EPR-Spektroskopie ist eine wertvolle Methode, um Abstände von 1.5 bis 10 nm zwischen zwei Spinmarkern zu messen. Diese Information kann für Strukturbestimmungen hilfreich sein, wo traditionelle Methoden wie Kristallstrukturanalyse und NMR nicht angewendet werden können. Zusätzlich ist es möglich, Änderungen in Konformation und Flexibilität zu verfolgen. Für diese Studien haben sich stabile Nitroxidradikale als Spinmarker etabliert. Diese werden spezifisch durch die site-directed spin labelling Methode (SDSL) kovalent an das zu untersuchende Biomolekül gebunden. In den letzten Jahren wurden weitere Spinmarker für Abstandsbestimmungen mittels EPR-Spektroskopie entwickelt. Besonders interessant sind Triarylmethylradikale (im Folgenden abgekürzt als Trityl) und paramagnetische Metallzentren.
Im Vergleich zu Nitroxidradikalen hat das Tritylradikal einige Vorteile: Eine höhere Stabilität in einer reduzierenden Umgebung wie im Inneren von Zellen, längere Elektronenspin-Relaxationszeiten bei Raumtemperatur und ein schmaleres EPR-Spektrum. Deswegen ist dieses organische Radikal ein alternativer Spinmarker, der besonders gut für die Forschung von Biomolekülen in einer nativen Umgebung unter physiologischen Bedingungen geeignet ist. Auch paramagnetische Metallzentren sind weniger reduktionsempfindlich als Nitroxidradikale. Zusätzlich sind diese Spinmarker interessant in biologischen Fragestellungen. Zum Beispiel besitzen zahlreiche Enzyme paramagnetische Manganzentren als Cofaktoren. Zudem kann Magnesium, ein wesentlicher Cofaktor in Enzymen, Nukleinsäuren und Nukleotid-Bindungsdomänen der G- und Membranproteine, oft durch das paramagnetische Mangan ersetzt werden. Um Abstandsmessungen an Biomolekülen, die nur ein Metallzentrum besitzen, durchzuführen, können zusätzliche Spinmarker in Form eines Nitroxid-, Tritylradikals oder eines anderen paramagnetischen Metallkomplexes mithilfe der SDSL-Methode kovalent gebunden werden.
Nitroxidradikale, Tritylradikale und Metallzentren haben deutlich unterschiedliche EPR-spektroskopische Eigenschaften, welche oft als orthogonale Spinmarker bezeichnet werden. Solche Spinmarker sind nützlich für die Untersuchung von verschiedenen Untereinheiten bei makromolekularen Komplexen. Somit können die intramolekularen Abstände innerhalb einer Untereinheit sowie intermolekularen Abstände zwischen den unterschiedlichen Untereinheiten mit nur einer einzigen Probe bestimmt werden. Zusätzlich können die orthogonalen Marker sehr effektiv genutzt werden, um Metallzentren in Biomolekülen mithilfe der Trilateration-Strategie genau zu lokalisieren.
Die hier vorliegende Doktorarbeit beschäftigt sich mit der Nutzung dieser neuen Spinmarker für Abstandsmessungen. Solche Spinmarker sind noch kaum erforscht, obwohl sie für biologische Anwendungen eine große Rolle spielen könnten.
Das erste Ziel dieser Doktorarbeit war eine Studie über Tritylradikale mithilfe der dipolaren EPR-Spektroskopie. Zu diesem Zweck wurden sowohl double quantum coherence (DQC) und single frequency technique for refocussing dipolar couplings (SIFTER) Experimente als auch Hochfrequenz pulsed electron electron double resonance (PELDOR) Experimente mit einem Trityl-Modellsystem durchgeführt. Dabei wurden die Besonderheiten der unterschiedlichen dipolaren Spektroskopiemethoden mit diesem Spinmarker untersucht, um die Empfindlichkeit und Robustheit für die Abstandsmessungen zu optimieren.
Das zweite Ziel war eine Studie über den Einfluss der Hochspin-Multiplizität des Mangans auf die Abstandsbestimmungen. Für diesen Zweck wurde zuerst ein Modellsystem mit einem orthogonalen Mn2+ Ion und Nitroxidradikal mithilfe der PELDOR-Spektroskopie untersucht. Anschließend wurde ein weiteres Modellsystem mit zwei Mn2+-Ionen untersucht, um PELDOR und relaxation-induced dipolar modulation enhancement (RIDME) Experimente bezüglich ihrer Empfindlichkeit und Robustheit sowie Genauigkeit der Datenanalyse zu optimieren.
Das Trityl-Modellsystem wurde in der Arbeitsgruppe von Prof. Sigurdsson synthetisiert. Die EPR Messungen wurden bei zwei verschiedenen Mikrowellenfrequenzen (34 und 180 GHz) durchgeführt. Es wurde gezeigt, dass die Auswahl der optimalen Methode von den EPR-spektroskopischen Eigenschaften des Systems bei den jeweiligen Mikrowellenfrequenzen abhängig ist. Das EPR-Spektrum des Trityls ist bei 34 GHz so schmal, dass das ganze Spektrum von einem üblichen Mikrowellenpuls angeregt werden kann. In diesem Fall sind die DQC und SIFTER Experimente am besten geeignet. Der mit diesen Methoden bestimmte Abstand von 4.9 nm ist in guter Übereinstimmung mit Werten aus der Literatur. Es wurde festgestellt, dass die SIFTER Messung eine höhere Empfindlichkeit als DQC besitzt, da das Signal-zu-Rausch Verhältnis um den Faktor vier größer ist. Außerdem ist die SIFTER-Methode experimentell weniger anspruchsvoll, da ein deutlich kürzerer Phasenzyklus für die Mikrowellenpulse benötigt wird. ...
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
The cardiovascular system (CVS) consists of heart and blood vessels, forming a close circulatory loop. All tissues depend on the nutrients and molecular oxygen (O2) delivered by the blood. Therefore, it is not surprising that the CVS is one of the first working systems and the heart is the first functional organ in the forming embryo (Baldwin 1996). The building blocks of blood vessels are endothelial cells (ECs), which form the endothelium, a specialized epithelium that defines the luminal surface of the vessels (Pugsley and Tabrizchi 2000). The process of blood vessel development comprises several steps. The first events occurring are the formation of new vessels de novo to constitute the primary vascular loop known as vasculogenesis. During vasculogenesis the vascular precursors, known as angioblasts, migrate and coalesce to form the axial vessels. Subsequently, the main vessels undergo a specification step where they acquire either arterial or venous identity. As the embryo increases in size, the main vascular loop needs to increase in complexity. In order to reach all the different parts of the developing organs, new blood vessels are formed from pre-existing ones, a phenomenon known as angiogenesis (Gore et al. 2012).
Mature blood cells have a short lifespan. Therefore, hematopoietic stem cells (HSCs) are required throughout lifetime to constantly form new blood cells in a process called hematopoiesis. Interestingly, endothelial and immune cells development have been shown to converge at different points during their development, one of which is developmental hematopoiesis. During embryogenesis, definitive hematopoiesis occurs in a tissue called hemogenic endothelium (HE), a specialized subset of ECs at the ventral wall of the dorsal aorta (DA). HE acquires hematopoietic potentials and gives rise to HSCs, through a process known as endothelial-to-hematopoietic transition (EHT). During EHT, these specialized ECs extrude from DA and colonize the so-called aorta-gonadmesonephros (AGM) region, forming the native HSCs (Paik and Zon 2010).
As vascular development requires different steps, the molecular pathways involved are many. The Notch signaling pathway has been demonstrated to be one of the main players in vascular development. Among other functions, Notch signaling has been shown to be important during EHT. In the murine model, Runx1, a master regulator of HSC formation, has been shown to be transcriptionally regulated by NOTCH1 through GATA2 activation. This observation was later corroborated by knockdown studies for notch1a and notch1b in zebrafish (Butko, Pouget, and Traver 2016). Another essential pathway for vascular development is the HIF pathway. Hif-1α, Hif-1β and Hif-2α mouse mutants show severe vascular defects that result in early embryonic lethality (Simon and Keith 2008), which hinders a deep analysis of the phenotypes incurring in the mutant embryos. In addition, deletion of Hif-1α specifically in myeloid cells showed abnormalities in the motility, invasiveness, and adhesion of macrophages (Cramer et al. 2003). Intriguingly, Hif-1α deletion in vascular endothelial cadherin-expressing cells led to a significant but partial reduction of HSC number, suggesting that other players may be involved in this pathway (Imanirad et al. 2014).
Zebrafish embryos have been shown to be tolerant to hypoxia at very early stages of development (Padilla and Roth 2001). Also, zebrafish embryos develop externally and this allows to finely manipulate the environment where they grow (Lieschke and Currie 2007). These features make zebrafish an ideal model to investigate how hypoxia and Hif transcription factors affect vertebrate vascular development. In this study, I will examine the impact of hypoxia on zebrafish vascular development. Specifically, I will dissect the role of hif-1α in macrophage-EC interactions during vascular development and repair. Moreover, I show redundant functions for hif-1α and hif-2α in HSC development upstream of Notch signaling.
Structural biology often employs a combination of experimental and computational approaches to unravel the structure-function paradigm of biological macromolecules. This thesis aims to approach this combination by the application of Pulsed Electron-Electron Double Resonance (PELDOR/DEER) spectroscopy and structural modelling. In this respect, PELDOR spectroscopy in combination with site-directed spin labelling (SDSL) of proteins is frequently used to gain distance restraints in the range from 1.8 to 8 nm. The inter-spin distance and the flexibility of the spin labelled protein domains are encoded in the oscillation and the dampening of the PELDOR signal. The intrinsic flexibility of the commonly used MTSSL (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) spin label itself can be an obstacle for structural modelling if the flexibility of the label is large compared to the flexibility of the protein domains. In this thesis the investigation of two multi-domain proteins by the 4-pulse PELDOR sequence is presented. At first, the N-terminal polypeptide transport-associated (POTRA) domains of anaOmp85, a rigid three domain protein, giving well-defined PELDOR distance restraints, is investigated. The experimental restraints are used for structure refinement of the X-ray structure and reveal a strong impact of the intrinsic flexibility of MTSSL on the accuracy of structural refinement. The second example, K48-linked diubiquitin, is a highly flexible multi-domain protein on which the flexibility of MTSSL is of minor impact on structural modelling. In this case, the distance restraints are utilized to determine conformational ensembles. Due to the high intrinsic flexibility already characterizing diubiquitin the recently developed 7-pulse Carr-Purcell (CP) PELDOR sequence was applied to investigate longer ubiquitin chains. This sequence enables to measure dipolar oscillations with an extended time window, allowing a good separation between inter- and intramolecular contributions even for long distance and broad conformational distributions, thereby providing an increased accuracy of the obtained distance distributions.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
Proteinen die ExHepatitis C ist eine entzündliche Erkrankung der Leber, die durch das Hepatitis-C-Virus (HCV) verursacht wird. Trotz vieler Bemühungen ist heutzutage immer noch keine prophylaktische Vakzinierung verfügbar. Neuartige Therapien versprechen eine hohe Heilungsrate, sind aber mit hohen Kosten verbunden. HCV induziert oxidativen Stress, welcher für das Auftreten und die Progression der Pathogenese eine zentrale Rolle spielt. Um zellulären Stress (z.B. durch ROS) entgegenzuwirken, haben Zellen cytoprotective und detoxifizierende Mechanismen entwickelt, die die zelluläre Homöostase aufrechterhalten. Dabei kontrolliert der redoxsensitive Transkriptionsfaktor Nrf2 als Heterodimer zusammen mit sMaf- pression von cytoprotective und ROS-detoxifizierenden Genen. Vorherige Studien haben gezeigt, dass HCV den Nrf2/ARE-Signalweg beeinträchtigt. Dabei induziert HCV eine Translokation der sMaf-Proteine aus dem Zellkern in das Cytoplasma, wo diese das virale Protein NS3 binden. Im Cytoplasma lokalisierte sMaf-Proteine verhindern dadurch eine Translokation von Nrf2 in den Zellkern. Folglich ist die Expression von Nrf2/ARE-abhängigen cytoprotective Genen inhibiert und intrazelluläre ROS-Spiegel dauerhaft erhöht. Ein weiterer zentraler cytoprotective Mechanismus ist die Autophagie. Sie dient der Aufrechterhaltung der zellulären Homöostase durch den Abbau von defekten Proteinen und Organellen. Des Weiteren ist bekannt, dass Autophagie nicht nur im Laufe von Nährstoffmangel induziert wird, sondern auch durch erhöhte Mengen an ROS. In sämtlichen Studien konnte beobachtet werden, dass Autophagie für die Aufrechterhaltung des viralen Lebenszyklus eine wesentliche Rolle spielt, da sie mit der Ausbildung des membranous web, der Translation, der Replikation und der Freisetzung des Virus interferiert. Ausgehend davon sollte in dieser Arbeit zunächst die Relevanz von HCV-induziertem oxidativen Stress, resultierend aus der Nrf2/ARE-Signalweginhibition, als möglicher Aktivator der Autophagie untersucht werden. Dabei wurde in HCV-positiven Zellen eine Akkumulation von LC3-II beobachtet, was auf eine Induktion der Autophagie schließen lässt. In Übereinstimmung damit wurde eine erhöhte Expression von Autophagie-Markerproteinen in HCV-infizierten PHHs detektiert. Im Laufe der Autophagie wird p62 abgebaut. Somit sollte eine Induktion der Autophagie in einer Verminderung der Menge an p62 resultieren. Nichtsdestotrotz ist eine Akkumulation von p62 in HCV-positiven Zellen nachzuweisen. Dies erscheint zunächst widersprüchlich. Aufgrund der Tatsache, dass die Expression der katalytischen Untereinheit des Proteasoms (PSMB5) Nrf2-abhängig ist, führt die beeinträchtigte Nrf2-Aktivität in HCV-positiven Zellen jedoch zu einer verringerten Aktivität des konstitutiven Proteasoms. Dieser Befund kann auch die erhöhte Halbwertzeit von p62 in HCV-positiven Zellen erklären. Kürzlich wurde ein Zusammenspiel des Nrf2/ARE-Signalwegs und der Autophagie beobachtet. Dabei kann Nrf2 nicht nur über den kanonischen Signalweg aktiviert werden, sondern auch durch eine direkte Interaktion des phosphorylierten Autophagie-Adaptorproteins p62 (pS[349] p62) mit Keap1. In HCV-positiven Zellen können nicht nur eine Zunahme der Gesamtmenge von p62 beobachtet werden, sondern auch erhöhte Mengen an pS[349] p62. Die Berechnung des Quotienten aus pS[349] p62 und p62 zeigt in etwa eine Verdopplung der Menge an pS[349] p62 , was auf eine vermehrte Phosphorylierung von p62 in HCV-positiven Zellen rückschließen lässt. Des Weiteren konnte beobachtet werden, dass erhöhte Mengen an ROS, wie sie auch in HCV-positiven Zellen vorkommen, Autophagie induzieren können, die durch eine Akkumulation von LC3-II und die Zunahme von LC3 Puncta charakterisiert ist. Auch eine Zunahme von pS[349] p62 konnte beobachtet werden. Ferner resultierte die Überexpression der phosphomimetischen Mutante (p62 [S351E]) in einer Akkumulation von LC3-II, was auf die Fähigkeit von pS[349] p62 rückschließen lässt, Autophagie zu induzieren. Eine Modulation der Autophagie mittels der Inhibitoren 3-Methyladenin und Bafilomycin führte zu einer inhibierten Freisetzung von infektiösen viralen Partikeln und unterstreicht damit, dass der Autophagie eine essentielle Bedeutung bei der Freisetzung viraler Partikel zukommt. Eine HCV-Infektion wird sowohl von erhöhten Mengen an ROS als auch von einer Induktion der Autophagie begleitet. Dementsprechend führte eine Verminderung des intrazellulären Radikalspiegels durch eine Inkubation mit den Radikalfängern PDTC und NAC zu geringeren Mengen an LC3-II und pS[349] p62. Dabei konnte auch eine Abnahme der freigesetzten infektiösen viralen Partikel beobachtet werden, was ein Zusammenspiel zwischen erhöhten Mengen an ROS, Induktion der Autophagie und Virusfreisetzung nahelegt. Vorschlag: Erhöhte Mengen an ROS werden durch eine Aktivierung des Nrf2/ARE-Signalwegs detoxifiziert und würden somit den zuvor beschriebenen viralen Mechanismus verhindern. HCV die Aktivierung Nrf2/ARE-regulierter Gene beeinträchtigt, wurde die Hypothese aufgestellt, dass in HCV-positiven Zellen dieser komplexe Mechanismus dazu dient, die Translokation des pS[349] p62-abhängig freigesetzte Nrf2 in den Zellkern zu verhindern. Das wiederum hat eine eingeschränkte Expression von Nrf2/ARE-abhängigen Genen und Detoxifizierung von ROS zur Folge. Um diese Hypothese experimentell zu untersuchen, wurden HCV-positive und negative Zellen cotransfiziert mit dem p62 Wildtyp (p62 [wt]), der p62 phosphomimetischen Mutante (p62 [S351E]) oder einem Kontrollplasmid in Kombination mit einem Reporterkonstrukt, welches die Nrf2-Aktivierung darstellt (OKD48). Während in HCV-negativen Zellen im Vergleich zum p62 [wt] eine Transfektion mit p62 [S351E] zu einer signifikanten Aktivierung des Nrf2-abhängigen Reportergens führt konnte dies in HCV-positiven Zellen nicht beobachtet werden. Zusammengenommen beschreiben diese Ergebnisse einen neuartigen Mechanismus wie HCV das Zusammenspiel zwischen dem Nrf2/ARE-Signalweg, erhöhten Mengen an ROS und Autophagie beeinflusst. Dabei übt HCV einen negativen Effekt auf den Nrf2/ARE-Signalweg aus, um dem pS[349] p62-abhängig freigesetzten Nrf2 zu entkommen. Folglich werden erhöhte Mengen an ROS aufrechterhalten, die eine Induktion der Autophagie ermöglichen, welche für die Freisetzung viraler Partikel essentiell ist.
The composition of cellular membranes is extremely complex and the mechanisms underlying their homeostasis are poorly understood. Organelles within a eukaryotic cell require a non-random distribution of membrane lipids and a tight regulation of the membrane lipid composition is a prerequisite for the maintenance of specific organellar functions. Physical membrane properties such as bilayer thickness, lipid packing density and surface charge are governed by the lipid composition and change gradually from the early to the late secretory pathway. As the endoplasmic reticulum (ER) is situated at the beginning of the cells secretory pathway, it has to accept and accommodate a great variety and quantity of secretory and transmembrane proteins, which enter the ER on their way to their final cellular destination. Secretory proteins can be translocated into the lumen of the ER co- or posttanslationally and membrane proteins are being inserted and released into the ER membrane. In the oxidative milieu of the ER-lumen, supported by a variety of chaperones, proteins can fold into their native form.
If the folding capacity of the ER-lumen is exceeded, an accumulation of mis- or unfolded proteins in the lumen of the ER occurs, consequently triggering the unfolded protein response (UPR). This highly conserved program activates a wide-spread transcriptional response to restore protein folding homeostasis. In fact, 7 – 8% of all genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) are regulated by the UPR. The mechanism underlying the activation of the UPR by protein folding stress has been investigated thoroughly in the last decades and many of its mechanistic details have been elucidated. Recently, it became evident that aberrant lipid compositions of the ER membrane, collectively referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism of this membrane-activated UPR, however, remained unclear.
This study focuses on the UPR in S. cerevisiae and characterizes the inositol requiring enzyme 1 (Ire1) as the sole UPR sensor in S. cerevisiae. Active Ire1 forms oligomers and, collaboratively with the tRNA ligase Rlg1, splices immature mRNA of the transcription factor HAC1, which results in the synthesis of mature HAC1 mRNA and the production of the active Hac1 protein, which binds to UPR-elements in the nucleus and activates the expression of UPR target genes. Here, the combination of in vivo and in vitro experiments is being used, which is supplemented by molecular dynamics (MD) simulations performed by Roberto Covino and Gerhard Hummer (MPI for Biophysics, Frankfurt), aiming to identify the molecular mechanism of Ire1 activation by lipid bilayer stress. This study focuses on the analysis of the juxta- and transmembrane region of Ire1. Bioinformatic analyses revealed a putative ER-lumenal amphipathic helix (AH) N-terminally of and partially overlapping with the transmembrane helix (TMH). This predicted AH contains a large hydrophobic face, which inserts into the ER membrane, forcing the TMH into a tilted orientation within the membrane. The resulting unusual architecture of Ire1’s AH and TMH constitutes a unique structural element required for the activation of Ire1 by lipid bilayer stress.
To investigate the function of the AH in the physiological context, different variants of Ire1 were produced under the control of their endogenous promoter and from their endogenous locus. The functional role of the AH was tested, by disrupting its amphipathic character by the introduction of charged residues into the hydrophobic face of the AH. The role of a conserved negative residue between the TMH and the AH (E540 in S. cerevisiae) was tested by substituting it by a unipolar, polar, or positively charged residue. These variants were intensively characterized using a series of assays:
This thesis provides evidence that the AH is crucial for the function of Ire1: Mutant variants with a disrupted (F531R, V535R) or otherwise modified AH (E540A) exhibited a lower degree of oligomerization and failed to catalyze the splicing of the HAC1 mRNA as the Wildtype control. Likewise, the induction of PDI1, a target gene of the UPR, was greatly reduced in mutants with a disrupted or defective AH. These data revealed an important functional role of the AH for normal Ire1 function.
An in vitro system was established to analyze the membrane-mediated oligomerization of Ire1. This system enabled the isolated functional analysis of the AH and TMH during Ire1 activation by lipid bilayer stress. A fusion construct, coding for the maltose binding protein (MBP) from Escherichia coli (E. coli), N-terminally to the AH and TMH of Ire1 was produced. The heterologous production in E. coli, the purification and reconstitution of this minimal sensor of Ire1 in liposomes was established as part of this study. To analyze the oligomeric status of the minimal sensor in different lipid environments, continuous wave electron paramagnetic resonance (cwEPR) spectroscopic experiments were performed. These experiments revealed that the molecular packing density of the lipids had a significant influence of the oligomerization of the spin-labeled membrane sensor: increasing packing densities resulted in sensor oligomerization. The AH-disruptive F531R mutant, in which the amphipathic character of the AH was destroyed, showed no membrane-sensitive changes in its oligomerization status.
Thus, the activation of Ire1 by lipid bilayer stress is achieved by a membrane-based mechanism. According to the current model, the AH induces a local membrane compression by inserting its large hydrophobic face into the membrane. As membrane thickness and acyl chain order are interconnected, this compression simultaneously results in an increased local disordering of lipid acyl chains. Supporting MD simulations performed by Roberto Covino and Gerhard Hummer revealed that the bilayer compression is significantly more pronounced in a densely packed lipid environment, than in a lipid environment of lower lipid packing density. Hence, the energetic cost of the local compression increases with the packing density of the membrane, but is compensated for by the oligomerization of Ire1. This minimization of energetic cost induced by the membrane deformation of Ire1 forms the basis for the activation of Ire1 by lipid bilayer stress.
Inhibition of midbrain dopamine (DA) neurons codes for negative reward prediction errors, and causally affects conditioning learning. DA neurons located in the ventral tegmental area (VTA) display two-fold longer rebound delays from hyperpolarizing inhibition in comparison to those in the substantia nigra (SN). This difference has been linked to the slow inactivation of Kv4.3-mediated A-type currents (IA). One known suppressor of Kv4.3 inactivation is a splice variant of potassium channel interacting protein 4 (KChIP4), KChIP4a, which has a unique potassium channel inactivation suppressor domain (KISD) that is coded within exon 3 of the KChIP4 gene. Previous ex vivo experiments from our lab showed that the constitutive knockout of KChIP4 (KChIP4 KO) removes the slow inactivation of IA in VTA DA neurons, with marginal effects on SN DA neurons. KChIP4 KO also increased firing pauses in response to phasic hyperpolarization in these neurons. Here I show, using extracellular recordings combined with juxtacellular labeling in anesthetized mice, that KChIP4 KO also selectively changes the number and duration spontaneous firing pauses by VTA DA neurons in vivo. Pauses were quantified with two different statistical methods, including one developed in house. No other firing parameter was affected, including mean frequency and bursting, and the activity of SN DA neurons was untouched, suggesting that KChIP4 gene products have a highly specific effect on VTA DA neuron responses to inhibitory input.
Following up on this result, I developed a new mouse line (KChIP4 Ex3d) where the KISD-coding exon 3 of KChIP4 is selectively excised by cre-recombinase expressed under the dopamine transporter (DAT) promoter, therefore disrupting the expression of KChIP4a only in midbrain DA neurons. I show that these mice have a highly selective behavioral phenotype, displaying a drastic acceleration in extinction learning, but no changes in acquisition learning, in comparison to control littermates. Computational fitting of the behavioral data with a modified Rescorla-Wagner model confirmed that this phenotype is congruent with a selective increase in learning from negative prediction errors. KChIP4 Ex3d also had normal open field exploration, novel object preference, hole board exploration and spontaneous alternation in a plus maze, indicating that exploratory drive, responses to novelty, anxiety, locomotion and working memory were not affected by the genetic manipulation. Furthermore semi-quantitative IHC revealed that KChIP4 Ex3d mice have increased Kv4.3 expression in TH+ neurons, suggesting that the absence of KChIP4a increases the binding of other KChIP variants, which known to increase surface expression of Kv4 channels.
Furthermore, in the course of my experimental study I identified that the most used mouse line where cre-recombinase is expressed under the DAT promoter (DAT-cre KI) has a different behavioral phenotype during conditioning in relation to WT littermate controls. These animals displayed increased responding during the initial trials of acquisition and delayed response latency extinction, consistent with an increase in motivation, which is in line with a decrease in DAT function.
I propose a working model where the disruption of KChIP4a expression in DA neurons leads to an increase in binding of other KChIP variants to Kv4.3 subunits, promoting their increased surface expression and increasing IA current density; this then increases firing pauses in response to synaptic inhibition, which in behaving animals translates to an increase in negative prediction error-based learning.
Introduction:
The evolutionary patterns of symbiotic organisms are inferred using cophylogenetic methods. Congruent phylogenies indicate cospeciation or host-switches to closely-related hosts, whereas incongruent topologies indicate independent speciation. Recent studies suggest that coordinated speciation is a rare event, and may not occur even in the highly specialized associations. The cospeciation hypothesis was mainly tested for free-living mutualistic associations, such as plant-pollinator interactions, and host-parasitic systems but was rarely tested on obligate, mutualistic associations involving intimate physiological interactions. Symbionts with lower partner selectivity may not experience coordinated speciation due to frequent switching of partners. On the other hand, symbionts with high partner selectivity may influence each other’s evolution owing to the highly interdependent lifestyles. Symbiont association patterns are also influenced by habitat and it has been proposed that symbiotic interactions are stronger in warm regions as compared to cooler regions (also referred as latitudinal gradient of biotic specialization). This hypothesis however, has recently been challenged and it has been suggested that a gradient of biotic specialization may not exist at all. Reliable species concepts are a prerequisite for understanding the association and evolutionary patterns of symbiotic organisms. The species concepts of many groups traditionally relied on the morphological species concept, which may not be adequate for distinguishing species due to the: i) homoplasious nature of morphological characters, an due to the inability to distinguish cryptic species. Thus phylogenetic species concept along with coalescent-based species delimitation approaches, which utilize molecular data for inferring species boundaries have been used widely for resolving taxonomic relationships. Lichens are obligatory symbiotic associations consisting of a fungal partner (mycobiont) and one or more photosynthetic partners, algae, and/or cyanobacteria (photobionts). I used the lichen forming fungal genus Protoparmelia as my study system, which consists of ~25-30 previously described species inhabiting different habitats, from the arctic to the tropics. This makes Protoparmelia an ideal system to explore the association and evolutionary patterns across different macrohabitats.
Objectives:
The objectives of this thesis were to 1. Elucidate the phylogenetic position of Protoparmelia within Lecanorales, and infer the monophyly of Protoparmelia; 2. Understand species diversity within Protoparmelia s.str. using coalescent-based species delimitation approaches; and 3. To identify the Trebouxia species associated with Protoparmelia using phylogenetic and species delimitation approaches and to infer the association and cophylogenetic patterns Protoparmelia and Trebouxia in different macrohabitats.
Results and discussion:
Chapter 1: Taxonomic position of Protoparmelia
In the first part of this study I explored the taxonomic position of Protoparmelia within the order Lecanorales. Overall this study included 54 taxa from four families, sequenced at five loci (178 sequences). I found Protoparmelia to be polyphyletic and sister to Parmeliaceae.
Chapter 2: Multilocus phylogeny and species delimitation of Protoparmelia spp.
In this part of the study, I identified and delimited the Protoparmelia species forming a monophyletic clade sister to Parmeliaceae i.e., Protoparmelia sensu stricto group, based on the multilocus phylogeny and coalescent-based species delimitation approaches. I included 18 previously described and three unidentified Protoparmelia species, which represents ~70% of the total described species, and 73 other taxa, sequenced at six loci. I found that the sensu stricto group comprised of 25 supported clades instead of 12 previously described Protoparmelia species. I tested the speciation probabilities of these 25 clades using species delimitation softwares BP&P and spedeSTEM. I found nine previously unrecognized lineages in Protoparmelia and I propose the presence of at least 23 species for Protoparmelia s.str., in contrast to the 12 described species included in the study.
Chapter 3: Association and cophylogenetic patterns of Protoparmelia and its symbiotic partner Trebouxia
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