Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv
250 Episoden
1 Minute
Soil bacteria are exposed to constant changes in temperature, moisture, and oxygen content. Additionally, they have to encounter different antimicrobial substances, which are produced by competing bacteria. Those agents often target the bacterial cell envelope, which is an essential structure composed of the cell wall and cell membrane. In order to counteract such life-threatening conditions, bacteria developed signal transducing systems to monitor their environment and to respond signal-specifically to any stress conditions, mostly by differential gene expression. Different principles of signal transducing systems have been evolved: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic function (ECF) sigma factors. Bacillus subtilis is a soil bacterium, which counteracts cell envelope stress by four different 2CSs (LiaSR, BceRS, PsdRS, and YxdJK) and at least three different ECF sigma factors (σX, σM, and σW). In the course of the present thesis, the LiaSR 2CS was investigated in detail. The LiaSR 2CS of B. subtilis is a cell envelope stress-sensing system that shows a high dynamic range of induction in response to cell wall antibiotics like bacitracin. It provides no resistance against its inducer molecules. Rather, it is a damage-sensing system that maintains the cell envelope integrity under stress conditions. The membrane-anchored histidine kinase (HK) LiaS and its cognate response regulator (RR) LiaR work together with a third protein, LiaF, which was identified as the inhibitor of the 2CS. Upon induction, the target promoter PliaI is induced by phosphorylated LiaR, leading to the expression of the liaIH-liaGFSR locus, with liaIH as being the most induced genes. In the first part of this thesis, the mechanisms of stimulus perception and signal transduction of the LiaFSR system were analyzed. Therefore, the native stoichiometry of the proteins LiaF, LiaS, and LiaR were determined genetically and biochemically with a resulting ratio of 18 to 4 to 1. We found out that maintaining this specific stoichiometry is crucial for the functionality of the LiaFSR system and thus a proper response to cell envelope stress. Changing the relative protein ratios by the overproduction of either LiaS or LiaR leads to a constitutive activation of the promoter PliaI. These data suggest a non-robust behavior of the LiaFSR system regarding perturbations of its stoichiometry, which stands in contrast to quantitative analyses of other well-known 2CSs. Furthermore, a HK-independent phosphorylation of the RR LiaR was observed. This happened in each case if the amount of LiaR exceeded those of LiaS, irrespective of the presence or absence of a stimulus. By using growth media supplied with different carbon sources, acetyl phosphate was identified as being the phosphoryl group-donor for LiaR under these conditions. Moreover, by performing a mutagenesis experiment, we obtained genetic evidence that LiaS is a bifunctional HK offering both a kinase and a phosphatase activity. In the second part of this thesis, the liaI promoter was used to generate a protein expression toolbox for the use in B. subtilis, referred to as the LIKE (from the German “Lia-kontrollierte Expression”) system. PliaI is a perfect candidate for driving recombinant protein expression. It is tightly regulated under non-inducing conditions showing no significant basal expression levels. Depending on the inducer molecule concentration, it is induced up to 1000-fold reaching a maximum already 30 minutes after addition of the inducer. Two expression vectors, an integrative and a replicative one, were constructed consisting of an alternative liaI promoter, which was optimized to enhance promoter strength. Additionally, different B. subtilis expression hosts were generated that possess liaIH deletions to prevent undesired protein production. The feasibility of the LIKE system was evaluated by using gfp and ydfG as reporter genes and bacitracin as inducer molecule. As a result, both proteins were successfully overproduced.
1 Minute
The peptidoglycan cell wall (CW) and the actin-like MreB cytoskeleton are the majordeterminants of cell morphology in non-spherical bacteria. Bacillus subtilis is a rod-shaped Grampositive bacterium that has three MreB isoforms: MreB, Mbl (MreB–like) and MreBH (MreBHomologue). Over the last decade, all three proteins were reported to localize in dynamic filamentous helical structures running the length of the cells underneath the membrane. This helical pattern led to a model where the extended MreB structures act as scaffolds to position CW-synthesizing machineries along sidewalls. However, the dynamic relationship between the MreB cytoskeleton and CW elongation complexes remained to be elucidated. Here we describe the characterization of the dynamics of the three MreB isoforms, CW synthesis and elongation complexes in live Bacillus subtilis cells at high spatial and temporal resolution. Using total internal reflection fluorescence microscopy (TIRFM) we found that MreB, Mbl and MreBH actually do not assemble into an extended helical structure but instead into discrete patches that move processively along peripheral tracks perpendicular to the long axis of the cell. We found similar patch localization and dynamics for several morphogenetic factors and CW-synthesizing enzymes including MreD, MreC, RodA, PbpH and PBP2a. Furthermore, using fluorescent recovery after photobleaching (FRAP), we showed that treadmilling of MreB filaments does not drive patch motility, as expected from the structural homology to actin. Blocking CW synthesis with antibiotics that target different steps of the peptidoglycan biosynthetic pathway stopped MreB patches motion, suggesting that CW synthesis is the driving force of patch motility. On the basis of these findings, we proposed a new model for MreB fuction in which MreB polymers restrict and orient patch motility to ensure controlled lateral CW expansion, thereby maintaining cell shape. To further investigate the molecular mechanism underlying MreB action, we next performed a site-directed mutagenesis analysis. Alanine substitutions of three charged amino 2 acids of MreB generated a B. subtilis strain with cell shape and growth defects. TIRFM analysis revealed that the mutated MreB protein displayed wild-type localization and dynamics, suggesting that it is still associated to the CW elongation machinery but might be defective in an interaction important for MreB morphogenetic function. Thus, this mutant appears as as a good candidate to start characterizing the interactions between the three MreB isoforms and components involved in CW elongation. It might also help to understand the function of components of theCWsynthetic complexes, and how they are coordinated to achieve efficient CW synthesis. Finally, to investigate how the integrity of the CW is maintained, we studied the localization and dynamics of the LiaIH-system, which i s t he t arget o f L iaRS, a t wo-component system involved in cell envelope stress response. We found that under stress conditions, when liaI and LiaH genes are expressed, the proteins form static complexes that coat the cell membrane. LiaI is required for the even distribution of the LiaH in the membrane. Taken together, these data suggest that LiaIH complexes may protect the cell from CW damage. Taken together, the findings described in this thesis provide valuable insights into the understanding of CW synthesis in B. subtilis, which may open new perspectives for the design of novel antimicrobial agents.
1 Minute
Dinophytes are unicellular eukaryotic algae that, together with their closest relatives, ciliates and apicomplexans, belong to the superphylum of alveolates. Some of them, namely calcareous dinophytes (Thoracosphaeraceae, Peridiniales), develop an immotile calcareous cell during their life history. They accumulate in the oceans’ sediments analogously to terrestrial seed banks. Although the diversity of calcareous dinophytes was investigated in several studies, only a few of them provide data from coastal waters and sediments. The main goal of this thesis was to record the diversity of calcareous dinophytes from marine environments using morphological, anatomical, taxonomical and evolutionary approaches. An essential part of the project was to establish living dinophyte cultures, assuring constant access to fresh material for morphological and molecular analysis (chapters 1-5). The morphological diversity of extant species as well as those described from the fossil record was documented (chapter 1), and the conflict between molecular and the morphological data was presented (chapter 1-5). Furthermore, detailed morphological descriptions of two new species, Scrippsiella bicarinata und S. kirschiae (chapter 2) and morphological analysis of Bysmatrum sp., a species of doubtful phylogenetic position (chapter 4), were provided. Scrippsiella trochoidea (basionym: Glenodinium trochoideum), a species with a previously ambiguous description, has been redescribed and epitypified by myself based on material collected from the type locality to assure a reliable determination of this species (Chapter 3). Comparative ultrastructure investigations using light and electron microscopic techniques at various stages of the life cycle showed that the anatomical structure during the biomineralization processes differs within subgroups of calcareous dinophytes (chapter 5) and could be used as a useful phylogenetic trait.
1 Minute
Das Epstein-Barr Virus (EBV) ist mit einer Reihe von lebensbedrohlichen Krankheiten assoziiert. Dazu zählen unter anderem Nasopharynxkarzinome, Hodgkin-Lymphome und lymphoproliferative Erkrankungen nach Organtransplantationen. Dennoch gibt es bisher keinen wirksamen Therapieansatz, der sich spezifisch mit der Rolle von EBV in diesen malignen Erkrankungen auseinandersetzt. Das latente Membranprotein 1 (LMP1) ist das primäre Onkogen von EBV und essenziell für die Transformation von B-Zellen durch das Virus. Für eine effiziente Transformation von Zellen ist die Aktivierung verschiedener zellulärer Signalwege durch LMP1 notwendig. LMP1 besitzt jedoch keine enzymatische Aktivität und die Induktion der Signalwege ist somit abhängig von der Rekrutierung verschiedener zellulärer Adapterproteine. Die Ausbildung der notwendigen Signalkomplexe wird über zwei C-terminale Aktivierungs-Regionen (CTAR1 und CTAR2) vermittelt. Verschiedene Mitglieder der Tumornekrosefaktor (TNF)-Rezeptor-assoziierten Faktoren (TRAF)-Protein-Familie spielen bei der Induktion der Signalwege durch diese beiden CTAR-Domänen eine zentrale Rolle. Nach grundlegenden Protein-Protein-Interaktionsstudien zwischen LMP1 und rekombinanten TRAF-Proteinen wurde hier die Interaktion zwischen TRAF2 und LMP1 als Zielstruktur für Inhibitoren vorgestellt. TRAF2 ist essenziell für die Aktivierung des NF-κB-Signalweges durch die CTAR1-Domäne und somit für das Überleben EBV-transformierter Zellen. Die Bindung von TRAF2 an LMP1 wurde biochemisch näher charakterisiert und die gewonnen Erkenntnisse verwendet, um ein System zu etablieren, mit dem Inhibitoren gegen den Komplex aus LMP1 und TRAF2 identifiziert werden können. Dieses ELISA-basierte System erfüllt die Anforderungen, die allgemein an hochdurchsatzfähige Systeme gestellt werden. In einem Pilotscreen einer Bibliothek mit Naturstoffen wurden Substanzen identifiziert, die die Bindung von TRAF2 an LMP1 in vitro inhibierten. Die potenteste Substanz inhibierte die Interaktion von TRAF2 und LMP1 mit einem IC50 von 8 µM in diesen in vitro Studien. Weiterhin zeigte diese Substanz eine spezifische biologische Wirkung auf die Vitalität von EBV-transformierten B-Zellen. Zusätzlich konnte in den Protein-Protein-Interaktionsstudien zwischen den verschiedenen TRAF-Proteinen und LMP1 erstmals eine direkte Bindung von TRAF6 an LMP1 gezeigt werden. Entgegen der bisherigen Modellvorstellung, nach der TRAF6 indirekt über Adapterproteine an LMP1 gebunden wird, konnte hier gezeigt werden, dass TRAF6 direkt an die LMP1-Sequenz P379VQLSY innerhalb der CTAR2-Domäne bindet. Diese Sequenz ist essenziell für die Aktivierung verschiedener TRAF6-abhängiger Signalwege durch die CTAR2-Domäne. Auf der Oberfläche von TRAF6 wird die Bindung an LMP1 durch dieselbe Bindetasche vermittelt, über die auch die Interaktion mit zellulären Rezeptoren stattfindet. Diese direkte Interaktion zwischen LMP1 und TRAF6 ist wichtig für die Aktivierung des NF κB-Signalweges durch die CTAR2-Domäne. TRAF6-Mutanten, die nicht mehr in der Lage waren, mit LMP1 zu interagieren, waren ebenfalls nicht mehr dazu fähig, die Induktion von NF κB-Signalen durch die CTAR2-Domäne von LMP1 in embryonalen TRAF6-/- Mausfibroblasten wiederherzustellen. Ebenfalls konnte neben der direkten Bindung von TRAF6 an LMP1 hier eine weitere neue Protein-Protein-Interaktion für TRAF6 beschrieben werden. TRAF6 bindet direkt an das TNF-Rezeptor-assoziierte Todesdomänenprotein (TRADD). Die Interaktion zwischen TRAF6 und TRADD unterscheidet sich jedoch von der Bindung anderer TRAF-Proteine an TRADD. Die in vitro Studien zeigten, dass TRAF6 in der Lage ist, sowohl mit Teilen des N-Terminus, als auch mit Teilen des C-Terminus von TRADD zu interagieren. Diese bisher nicht beschriebene Art der direkten Interaktion von TRAF6 mit TRADD eröffnet neue Einblicke in den Aufbau des LMP1-Signalkomplexes.
1 Minute
Morbus Alzheimer ist die am weitesten verbreitete Form der Demenz mit verheerendem Einfluss auf das Leben der Betroffenen, deren Angehörigen sowie die weltweiten Sozialsysteme. Durch die alternde Gesellschaft gewinnt die Krankheit immer mehr an Relevanz, vor allem da bisher weder die Ursache der Erkrankung bekannt noch hinreichende Therapien etabliert sind. Ein zentrales Problem in der Entwicklung neuer Therapieansätze liegt in dem Fehlen eines verlässlichen, breit einsetzbaren Frühdiagnoseverfahrens. In der vorliegenden Arbeit sollte geklärt werden, ob ein Nachweis der Alzheimer-typischen Proteinaggregate von Aβ und Tau in der Retina für ein potentielles Diagnoseverfahren genutzt werden kann. Dabei sollte eine Methode etabliert werden, bei dem eine Aβ- und Tau-spezifische, fluoreszente Sonde systemisch appliziert und schließlich das gebundene Fluorophor unter Verwendung eines Retina-Scanners detektiert wird. Dieses Verfahren sollte zunächst in Mausmodellen des Morbus Alzheimer etabliert werden, die laut Literaturangaben Aβ-Plaques (u.a. DeltaE9 und Tg2576) bzw. fibrilläre Tau-Aggregate (P301S) im Gehirn und der Retina entwickeln. Unter Verwendung Aβ- bzw. Tau-spezifisch bindender Fluorophore (FSB und BSc4090) konnte in vivo kein Nachweis von Aβ-Plaques in der Retina mehrerer transgener Mauslinien erbracht werden. Begleitende histologische Untersuchungen zeigten ebenfalls keinerlei Anzeichen für Aβ-Plaques in diesen Retinae, was im Widerspruch zu publizierten Arbeiten steht. In der P301S-Linie gelang hingegen erstmals der in vivo Nachweis von fibrillären Tau-Aggregaten in retinalen Ganglienzellen unter Verwendung des Farbstoffes FSB. Dieser Befund wurde genutzt, um den Verlauf der Pathologie in der Retina der P301S-Linie über mehrere Monate zu verfolgen. Dabei wurde ein kontinuierlicher Anstieg der Anzahl von retinalen Ganglienzellen mit fibrillären Tau-Aggregaten festgestellt. Anzeichen für einen hierdurch bedingten Zellverlust fanden sich allerdings nicht. Diese Methode erlaubte erstmalig die Durchführung nicht-invasiver Langzeitexperimente zur Untersuchung von Wirkstoffen, die eine Bildung fibrillärer Tau-Aggregate verhindern sollen. Mit den verwendeten GSK3-Inhibitoren konnte allerdings kein Effekt beobachtet werden. Parallel zu den Experimenten im Mausmodell wurden histologische Untersuchungen an den Retinae verstorbener Alzheimer-Patienten durchgeführt. Analog zu den Befunden in den Mausmodellen konnten in den Retinae von Alzheimer-Patienten keine Aβ-Plaques nachgewiesen werden. Zwar wurden Ablagerungen von hyperphosphoryliertem Tau in den Retinae der Alzheimer-Fälle nachgewiesen, fibrilläre Tau-Aggregate fanden sich allerdings nicht. Somit kann der in vivo Nachweis fibrillärer Tau-Aggregate in der Retina des P301S-Modells nicht auf die humane Diagnose übertragen werden
1 Minute
In the visual system of Drosophila, photoreceptor (R) neurons elaborate a precise retinotopic map of visual space in the brain. The retina consists of 750 ommatidia, each containing eight photoreceptor subtypes (R1-R8). R1-6 axons terminate in the first optic ganglion, the lamina. R7 and R8 axons extend through the lamina to innervate the second optic ganglion, the medulla. To maintain retinotopy in the lamina, R1-R6 photoreceptor axons have to undergo a complex axonal sorting during development, a process called neural superposition. The mechanisms responsible for the establishment of the highly organized connection pattern needed for retinotopy remain incompletely understood. The transmembrane receptor Golden goal (Gogo) is a known regulator of the developing Drosophila visual system. During R8 pathfinding, Gogo acts in two distinct steps. In larvae Gogo mediates repulsive axon-axon interactions between R8 axons in the medulla to maintain proper spacing. During pupal development, Gogo is required in R8 axons for afferent-target interactions to promote layer recognition. The aim of this thesis is to study how Gogo regulates target selection of R1-R6 axons in the lamina to increase our knowledge on how target specificity is controlled in vivo. The present work shows that Gogo is required for R1-R6 axon lamina targeting and target cartridge selection in distinct developmental steps. To analyze the consequences of loss of gogo function specifically in photoreceptor cells, I generated genetic mosaic eyes using targeted mitotic recombination. During larval and early pupal development loss of gogo function in large clones of R axons results in a disruption of R1-R6 fascicle pattern formation across the lamina plexus. Using single photoreceptor type rescue, I provide evidence that the first outgrowing axon R8 uses Gogo to identify its intermediate target in the lamina and to function as a pioneer axon for all follower R1-R6 axons for their correct patterning along the lamina plexus. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Thus, Gogo acts in a partially redundant fashion with local guidance cues provided by neighboring axons. Additionally, during pupal stages at the onset of photoreceptor sorting, I further show that R1-R6 axons fail to choose correct target cartridges in the lamina when Gogo is absent in a large fraction of R cells. I show that gogo mutant R1-R6 axons target correctly to wild-type areas, whereas wild-type R1-R6 axons fail to project correctly to areas innervated by mutant R axons. Interestingly, rescue of Gogo in R8 axons was not only sufficient for fascicle patterning earlier in development but also for R1-R6 axons to select their proper target cartridges in the lamina during neural superposition. Finally, in a third developmental step, Gogo is required for the elongation of R1-R6 axons along lamina cartridges within the neuropile. In the absence of Gogo axons fail to elongate in parallel fashion and intermingle with mutant axons of neighboring cartridges. This suggests that Gogo, similar to its role in medulla targeting, permits photoreceptor axons to stay separated from each other. Based on the results of this thesis, I propose that Gogo contributes to retinotopic map formation in the Drosophila lamina during three steps: initial target recognition of R1-R6 fascicles, target cartridge selection and cartridge elongation.
1 Minute
1 Minute
The ability to adapt to environmental changes, to learn and to memorize information is one of the brain’s most extraordinary features. One important process underlying this ability is considered to be synaptic plasticity, i.e. the structural and functional modification of synaptic connections. Synaptic plasticity can occur either by genesis or elimination of synaptic connections, or at existing connections by modifications in the strength of synaptic transmission. Synaptic connections are complex entities consisting of different functional structures: The majority of hippocampal and cortical excitatory synapses are made up of a postsynaptic compartment called dendritic spine and a presynaptic compartment called bouton. Within the spine and the bouton dense molecular structures, which serve the synaptic transmission between pre‐ and postsynapse, exist, namely the postsynaptic density (PSD) in the spine, and the active zone (AZ) in the bouton. All these structures are correlated in size and with synaptic strength. The function of this correlation serves the efficient and fast transmission of neuronal signals. During synaptic plasticity, a coordinated change in the size of all synaptic structures is expected, for the maintenance of their correlation. However, to date, such coordinated modifications have not been examined in detail. Furthermore, the mechanisms underlying the maintenance of structural and functional changes after synaptic plasticity remain poorly understood. The aim of this thesis was to explore these questions. To achieve this I carried out two complementing experimental approaches: In a first set of experiments, I studied changes in spine and PSD size by twophoton time‐lapse imaging to explore correlated modifications in these two synaptic structures. To induce structural spine plasticity I stimulated single dendritic spines of Schaffer collateral synapses in cultured hippocampal slices by two‐photon glutamate uncaging. This was shown previously to be accompanied by an increase in spine size and synaptic strength. To visualize structural plasticity of spines and their PSD, the cytosolic marker tdTomato and EGFP‐tagged structural proteins of the PSD, namely PSD‐95 and Homer1c, were co‐expressed. PSD‐95 and Homer1c are important and abundant scaffolding proteins of the PSD, which have been used previously as markers for PSD size. I found that both PSD‐95 and Homer1c levels increased after spine stimulation. Homer1c increased rather rapidly whereas PSD‐95 did so in a delayed manner relative to the increase in spine volume. Thus, the naïve correlation between PSD protein level and spine volume was only transiently disrupted after plasticity induction, but was reestablished over a time course of 3 hours. Furthermore, PSD‐95 level only increased significantly in spines with persistent enlargement, but not in spines with non‐persistent enlargement. On the other hand, Homer1c level initially increased both in spines with and without persistent enlargement, and then decayed back to original level in spines with non‐persistent enlargement. Because the increase in PSD‐95 level was delayed, I investigated whether the application of the PKA activator forskolin, which supports an increased and persistent enlargement of spines after glutamate uncaging, might promote and therefore accelerate an increase in PSD‐95 level. However, these experiments led to unexpected results: forskolin application neither had an effect on spine volume nor on PSD‐95 level increase. Although PSD‐95 and Homer1c are important and abundant PSD scaffolding proteins, they represent only two out of a multitude of proteins which form the PSD. Consequently, an increase in the PSD marker proteins does not necessarily represent an increase of the PSD as a whole. Therefore, in a second experimental approach, I applied electron microscopy to stimulated spines which displayed a stable enlargement over 3 hours after stimulation. Hereby, I was able not only to reconstruct the spine and the entire PSD, but also the bouton at the stimulated spine: I found that spine, PSD and bouton displayed matching dimensions 3 hours after stimulation, similar to naïve, unstimulated spines. In summary, by combining two‐photon glutamate uncaging with time‐lapse imaging and electron microscopy, I found that spine, the PSD and bouton increase during structural plasticity, and that the correlation between these structures is reestablished after stimulation on a time scale of 3 hours. Furthermore, an increase of synaptic structures correlates with the stabilization of synaptic modifications after plasticity. This suggests a model where the balancing of synaptic structures is a hallmark for the stabilization of structural modifications during synaptic plasticity.
