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The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.
Life and biological resilience rely on the execution of precise gene expression profiles. A key mechanism to ensure cellular homeostasis is the regulation of protein synthesis. Recent studies have unveiled an intrinsic regulatory capacity of ribosomes, previously considered mere executors of mRNA translation. Neurons in particular finely regulate protein synthesis, at both global and local levels. This sustains their complex morphology and allows them to rapidly transmit, integrate, and respond to external stimuli. In this thesis, I investigated the neuronal ribosome and how subcellular environments and physiological perturbations shape it, by profiling its molecular composition, functional interconnections, and cellular distribution.
First, I used genetic engineering, biochemical purification, and mass spectrometry, to characterize in an unbiased manner the translation machinery specifically from excitatory and inhibitory neurons of the mouse cortex. I found that neuronal ribosomes commonly interact with RNA-binding proteins, components of the cytoskeleton, and proteins associated with the endoplasmic reticulum and vesicles. In line with the requirement for local protein synthesis in the distal parts of neurons, we observed that neuronal ribosomes preferentially interact with proteins involved in cellular transport. Remarkably, I observed a strong association between ribosomes and pre-synaptic vesicles, which suggests a potential regulatory interaction between local translation and neuronal activity.
Intriguingly, I and others have observed mRNAs encoding for core ribosomal proteins (RPs) among the genes most enriched in neuronal processes. This observation challenges two historical assumptions of ribosome biology: (1) new RPs are incorporated only into newly forming ribosomes, and (2) this incorporation occurs only in the nucleus and perinuclear region. In my PhD, I aimed to directly test these two assumptions and if proven wrong ask whether and why neurons would localize RP mRNAs far from their known assembly site.
Employing a combination of metabolic labeling and highly sensitive mass spectrometry techniques, I discovered that a subset of RPs rapidly and dynamically binds on and off mature ribosomes. Strikingly, this incorporation does not depend on the supply of new ribosomes from the nucleus. Therefore, my data refuted the assumption that ribosomes are built and degraded as a unit and revealed a more dynamic view of these machines, which can actively exchange core components. In particular, I found that the association of certain exchanging RPs is influenced by location (e.g., cell body versus neurites) and cellular state (e.g., post-oxidative stress). Neurons may use this mechanism to repair and/or specialize their protein synthesis machinery in a rapid and context-dependent manner.
Finally, I asked whether some steps of ribosome biogenesis could also take place in distal processes. Although most steps of ribosome assembly occur within the nucleus, the final stages of maturation are known to occur in the cytosol. By combining several imaging and biochemical approaches, I found that cytosolic (but not nuclear) pre-ribosomal particles are present in neuronal processes. Through the incorporation of new RPs into these immature particles, neurons may be able to locally “turn on” previously incompetent ribosomes. This may enable regions near synapses to enhance and customize their translational capacity, independently of the central pool of ribosomes from the cell body. Indeed, I observed that synaptic plasticity induces a maturation of cytosolic pre-ribosomes.
In summary, this thesis shows how neuronal ribosomes can sense cellular states, respond by adjusting their core composition, and in doing so influence the local capacity for protein synthesis. By overturning long-held assumptions in ribosome biology, this work highlights new molecular mechanisms of gene expression and enriches our understanding of the rapid and dynamic strategies cells employ to operate, thrive, and adaptively respond to environmental changes.
Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.
Die Biologie der Onkogene
(1991)
Onkogene wurden zuerst als dominante, maligne transformierende Gene in den Genomen von Retroviren identifiziert. In normalen Zellen existieren eng verwandte Gene, die phylogenetisch hochkonserviert sind und wichtige regulatorische Funktionen bei Differenzierung und Wachstum haben. Diese Gene werden als Proto-Onkogene bezeichnet. Onkogene haben häufig ähnliche, Jedoch aberrante enzymatische oder regulatorische Aktivitäten wie die normalen Gene. Sie werden in verschiedene Klassen eingeteilt (Proteinkinasen, GTP-bindende Proteine, Wachstumsfaktor-Analoga, nukleare Proteine, Wachstumsfaktor-Rezeptor-Analoga). Die normalen Funktionen der Proto-Onkogene werden schon ansatzweise verstanden. So regeln beispielsweise die Analoga der Tyrosinkinase-Onkogene unter anderem die Hämatopoese. Ein neuer Vertreter dieser Gruppe wurde von uns isoliert und als c-tkl bezeichnet.
Onkogene treten in malignen Geweben auch unabhängig von Retroviren auf. Mit DNA aus Tumoren gelang es in Transfektionsexperimenten, normale Zellen zu transformieren. Die Analyse zeigte, daß ein Onkogen in die Zellen eingeschleust worden war, das man schon von Experimenten mit Retroviren kannte. Gene, die das Transformations-Potential der Onkogene inhibieren, werden als Tumor-Suppressor-Gene bezeichnet und stellen einen neuen Zweig der Krebsforschung dar.
Insgesamt ist die Entstehung einer Krebszelle immer an eine oder mehrere genetische Veränderungen gebunden, die ein regulatorisch wichtiges normales Gen funktionell ändern oder es zur falschen Zeit, in der falschen Zelle oder in der falschen Menge exprimieren. Diese genetische Änderung kann angeboren sein, sie kann durch die Infektion mit Viren, durch Strahlen, durch chemische Karzinogenese oder durch spontane Mutation erfolgen. Die Tatsache, daß heute eine Vielzahl von Genen bekannt ist, deren Veränderung Krebs induziert, läßt für die Zukunft eine wesentlich verbesserte Tumor-Diagnostik und eine spezifischere Therapie erwarten. Die systematische Aufklärung der Genome höherer Organismen dürfte für das Studium der Tumor-Suppressorgene und der Onkogene neue Erkenntnisse liefern.
Precise regulation of gene expression networks is required to develop and maintain a healthy organism before and after birth and throughout adulthood. Such networks are mostly comprised of regulatory proteins, but meanwhile many long non-coding transcripts (lncRNAs) are shown to participate in these regulatory processes. The functions and mechanisms of these lncRNAs vary greatly, however they are often associated with transcriptional regulation. Three lncRNAs, namely Sweetheart RNA (Swhtr), Fetal-lethal noncoding developmental regulatory RNA / Foxf1 adjacent non-Coding developmental regulatory RNA (Fendrr) and lncFsd2, were studied in this work to demonstrate the variety of cellular and biological processes that require lncRNA-mediated fine-tuning, in regard to the cardiopulmonary system.
Swhtr was found to be expressed exclusively in cardiomyocytes and became critical for regeneration after myocardial injury. Mice lacking Swhtr did not show issues under normal conditions, but failed to undergo compensatory hypertrophic remodeling after injury, leading to increased mortality. This effect was rescued by re-expressing Swhtr, demonstrating importance of the RNA. Genes dependent on Swhtr during cardiac stress were found to likely be regulated by NKX2-5 through physical interaction with Swhtr. Fendrr was found to be expressed in lung and interacted with target promoters through its RNA:dsDNA binding domain, the FendrrBox, which was partially required for Fendrr function. Fendrr, together with activated WNT signaling, regulated fibrosis related target genes via the FendrrBox in fibroblasts. LncFsd2, an ubiquitously expressed lncRNA, showed possible interaction with the striated muscle specific Fsd2, but its exact function and regulatory role remain unclear in muscle physiology. Immunoprecipitation and subcellular fractionation experiments suggest that lncFsd2 might be involved in nuclear retention of Fsd2 mRNA, thus fine-tuning FSD2 protein expression. These investigations have shed light on the roles of these lncRNAs in stress responses, fibrosis-related gene regulation, and localization processes, advancing our understanding of cardiovascular and pulmonary maintenance, reaction to injury, and diseases. The diverse and intricate roles of these three lncRNAs highlight how they influence various cellular processes and disease states, offering avenues for exploring lncRNA functions in different biological contexts.
All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
(2023)
Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.
Mitochondria perform essential energetic, metabolic and signalling functions within the cell. To fulfil these, the integrity of the mitochondrial proteome has to be preserved. Therefore, each mitochondrial subcompartment harbours its own system for protein quality control. However, if the capacity of mitochondrial chaperones and proteases is overloaded, mitochondrial misfolding stress (MMS) occurs. Upon this stress condition, mitochondria communicate with the nucleus to increase the transcription of nuclear encoded mitochondrial chaperones and proteases. This proteotoxic stress pathway was termed the mitochondrial unfolded protein response (UPRmt) aiming at restoring protein homeostasis. Despite being discovered over 25 years ago, the signalling molecules released by stressed mitochondria as well as the corresponding receptor and transcription factor remain poorly understood. With this study, we aimed at characterising the underlying signalling events and mechanisms of how mitochondria react to misfolded proteins. First, we aimed to establish different methods to induce MMS that triggers the transcriptional induction of mitochondrial chaperones and proteases detected by quantitative polymerase chain reaction. We were able to induce UPRmt signalling by overexpression of an aggregation-prone protein and by knock-down or inhibition of mitochondrial protein quality control components. To study the signalling in a time-resolved manner, we focused on the usage of the mitochondrial HSP90 inhibitor GTPP and the mitochondrial LONP1 protease inhibitor CDDO.
Early time point RNA sequencing analysis of cells stressed with GTPP or CDDO revealed upregulated genes in response to oxidative stress. Indeed, measurements of mitochondrial superoxide with the fluorescent dye MitoSOX showed increased levels of reactive oxygen species (ROS) upon MMS induction. In contrast, there was no induction of mitochondrial chaperones and proteases when combining MMS with antioxidants. Compartment-specific targeting of the hydrogen peroxide sensor HyPer7 revealed increased ROS levels in the intermembrane space and matrix of mitochondria, followed by elevated ROS levels in the cytosol at later time points. The importance of cytosolic ROS for the signalling was supported by preventing UPRmt induction with an inhibitor blocking the outer mitochondrial membrane pore. Thus, ROS were identified as an essential UPRmt signal.
To understand which cytosolic factor is modified by ROS, redox proteomics was performed. Here, reversible changes on cysteine residues of the HSP40 co-chaperone DNAJA1 were observed upon MMS. Consequently, transcriptional induction of UPRmt genes was abolished by DNAJA1 knock-down. To understand the function of DNAJA1 during UPRmt signalling, quantitative interaction proteomics upon MMS revealed an increased binding to mitochondrial proteins and its interaction partner HSP70. Immunoprecipitation confirmed a ROS-dependent interaction between HSP40 and HSP70. Increased binding to mitochondrial proteins represented a cytosolic interaction of DNAJA1 with mitochondrial precursor proteins, whose accumulation was confirmed by western blot. Moreover, a fluorescent protein targeted to mitochondria accumulated in the cytosol during GTPP treatment, confirming a reduced import efficiency upon MMS. Preventing the accumulation of precursors by a translation inhibitor or depletion of a general mitochondrial transcription factor resulted in reduced UPRmt activation. Thus, DNAJA1 is essential for UPRmt signalling, since its oxidation by mitochondrial ROS and its enhanced recruitment to mitochondrial precursors allows the integration of both MMS-induced signals.
To link these findings to an increased transcription of mitochondrial chaperones and proteases, we screened for transcription factors accumulating in the nucleus upon MMS by cellular fractionation mass spectrometry. We demonstrated that specifically HSF1 accumulates in nuclei of cells stressed with GTPP or CDDO. Depletion of HSF1 by knock-down or knock-out resulted in the abrogation of the UPRmt-specific transcriptional response. HSF1 activation was visualised by nuclear accumulation on western blot, a process inhibited by ROS and precursor suppression. Moreover, DNAJA1 depletion prevented HSF1 activation. Ultimately, we proved by immunoprecipitation that the inhibitory interaction between HSF1 and HSP70 is reduced upon MMS.
Thus, we conclude that MMS increases mitochondrial ROS that are released into the cytosol. In addition, the import efficiency is reduced upon MMS, resulting in the accumulation of non-imported mitochondrial precursor proteins in the cytosol. Both signals are recognised via DNAJA1 oxidation and substrate binding. The concurrent recruitment of HSP70 to DNAJA1 results in the loss of the inhibitory HSP70-HSF1 interaction. Thus, active HSF1 can migrate to the nucleus to initiate transcription of mitochondrial chaperones and proteases. These findings are in accordance with observations in yeast, where mistargeted mitochondrial proteins activate cellular stress responses. Our results highlight a surprising interconnection and dependence of the mitochondrial and the cytosolic proteostasis network, in which the UPRmt is activated by a combination of two mitochondria-specific proteotoxic stress signals.
This dissertation constitutes a series of successive research papers, starting with the characterization of various optogenetic tools up to the establishment of purely optical electrophysiology in living animals.
Optogenetics has revolutionized neurobiology as it allows stimulation of excitable cells with exceptionally high spatiotemporal resolution. To cope with the increasing complexity of research issues and accompanying demands on experimental design, the broadening of the optogenetic toolbox is indispensable. Therefore, one goal was to establish a wide variety of novel rhodopsin-based actuators and characterize them, among others, with respect to their spectral properties, kinetics, and efficacy using behavioral experiments in Caenorhabditis elegans. During these studies, the applicability of highly potent de- and hyperpolarizers with adapted spectral properties, altered ion specificity, strongly slowed off-kinetics, and inverted functionality was successfully demonstrated. Inhibitory anion channelrhodopsins (ACRs) stood out, filling the gap of long-sought equivalent hyperpolarizing tools, and could be convincingly applied in a tandem configuration combined with the red-shifted depolarizer Chrimson for bidirectional stimulation (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES). A parallel study aimed to compare various rhodopsin-based genetically encoded voltage indicators (GEVIs) in the worm: In addition to electrochromic FRET-based GEVIs that use lower excitation intensity, QuasAr2 was particularly convincing in terms of voltage sensitivity and photostability in C. elegans. However, classical optogenetic approaches are quite static and only allow perturbation of neural activity. Therefore, QuasAr2 and BiPOLES were combined in a closed-loop feedback control system to implement the first proof-of-concept all-optical voltage clamp to date, termed the optogenetic voltage clamp (OVC). Here, an I-controller generates feedback of light wavelengths to bidirectionally stimulate BiPOLES and keep QuasAr’s fluorescence at a desired level. The OVC was established in body wall muscles and various types of neurons in C. elegans and transferred to rat hippocampal slice culture. In the worm, it allowed to assess altered cellular physiology of mutants and Ca2+-channel characteristics as well as dynamical clamping of distinct action potentials and associated behavior.
Ultimately, the optogenetic actuators and sensors implemented in the course of this cumulative work enabled to synergistically combine the advantages of imaging- and electrode-based techniques, thus providing the basis for noninvasive, optical electrophysiology in behaving animals.
Die Bande q23 auf Chromosom 11 ist in zahlreiche reziproke chromosomale Translokationen verwickelt. Diese sind dominant mit dem Krankheitsphänotyp einer AML, ALL und seltener mit malignen Lymphomen und myelodysplastischen Syndromen assoziiert. Mittlerweile sind fünfundachtzig cytogenetische Aberrationen der Bande 11q23 bekannt. Das auf 11q23 betroffene Gen wird als das Mixed Lineage Leukemia (MLL), Acute Lymphoblastic Leukemia (ALL-1), Human Homolog of trithorax (HRX) oder als Human Trithorax 1 (Htrx1) bezeichnet. Die häufigsten Partnergene des MLL sind AF4 (40 %), AF9 (27 %), sowie ENL, AF6, ELL und AF10 (4-7 %).
Die Bruchpunkte von t(11;V) Translokationen sind nicht gleichmäßig über das gesamte, 92 kb große humane MLL-Gen verteilt, sondern liegen alle in der 8,3 kb großen Bruchpunktsregion Bpr. Auch innerhalb der Bpr ist die Verteilung der Translokationsbruchpunkte nicht homogen. Die Bruchpunkte von Patienten mit de novo Leukämien und einem Alter über einem Jahr liegen mehrheitlich in der 5’-Hälfte der Bpr, dem Subcluster I. Dagegen liegen die Bruchpunkte von Patienten mit therapiebedingten Leukämien und einem Alter unter einem Jahr überwiegend in der 3’-Hälfte der Bpr, dem Subcluster II.
Neuere Forschungsergebnisse zeigten, daß DNA-Doppelstrangbrüche auf zwei verschiedenen Chromosomen eine hinreichende Voraussetzung für das Entstehen chromosomaler Translokationen sind. Aufgrund der inhomogenen Verteilung der Translokationsbruchpunkte im MLL-Gen stellte sich die Frage, ob bestimmte Regionen dieses Gens für DNA-Doppelstrangbrüche prädisponiert sind. Interessanterweise ist Subcluster II extrem sensitiv gegenüber DNA-Doppelstrangbrüchen, die durch cytotoxische Agenzien oder Apoptose-auslösende Ereignisse induziert werden können. In unserer Arbeitsgruppe konnte eine etwa 200 bp große Region lokalisiert werden, über die sich nahezu alle Etoposid-induzierten DNA-Doppelstrangbrüche verteilten.
In dieser Arbeit konnte gezeigt werden, daß die Bildung von DNA-Doppelstrangbrüchen in dieser Region durch die Gabe eines Caspase-Inhibitors gehemmt werden kann. Eine Etoposid-induzierte Protein-DNA-Wechselwirkung konnte allerdings nicht nachgewiesen werden. In der Literatur fanden sich Hinweise darauf, daß Subcluster II im Gegensatz zu Subcluster I eine verstärkte Histonacetylierung aufweist. Basierend auf diesen Hinweisen sollte die Arbeitshypothese untersucht werden, ob Subcluster II einen geninternen Promotor des MLL-Gens darstellt.
Die potentielle Promotorregion wurde zunächst durch Computeranalysen eingegrenzt. Mit RT-PCR Experimenten wurde anschließend der potentielle geninterne Promotor des murinen Mll-Gens in einer murinen Fibroblastenzellinie lokalisiert, die einen Transkriptionsstop und eine Polyadenylierungssequenz in Exon 4 des Mll-Gens trug. Um die am Mausmodell gewonnenen Erkenntnisse auch im humanen System zu überprüfen, wurde die geninterne Promotorregion des humanen MLL Gens vor ein Luciferasereportergen kloniert. Durch RTPCR konnte der geninterne Transkriptionsstart im Subcluster II des humanen MLL-Gens lokalisiert werden. Damit konnte zum ersten Mal gezeigt werden, daß Transkriptionsinitiation und genetische Instabilität im Subcluster II des humanen MLL-Gens kolokalisieren.
Durch Deletionsmutanten wurde die Bedeutung der einzelnen Module dieser Promotorregion ermittelt. Dabei zeigte sich, daß die Anwesenheit von zwei retromobilen Elementen eine Enhancer-Funktion haben. Demgegenüber zeigte die homologe murine Sequenz, die in unserer Arbeitsgruppe gleichzeitig von S. Scharf untersucht wurde und für die keine erhöhte Anfälligkeit für DNA-Doppelstrangbrüche bekannt ist, nur eine schwache Promotoraktivität. Dies weist auf einen Zusammenhang zwischen der genetischen Instabilität von Subcluster II und der Rate geninterner Transkriptionsinitiationsprozesse hin.
Das Protein, für das das Transkript des geninternen murinen Promotors kodiert, wurde mittels immunhistologischer und Western Blot Experimente nachgewiesen. Dabei konnte gezeigt werden, daß dieses Protein, wie auch das MLL-Protein, proteolytisch durch Taspase1 und daß sich ein Mini-MLL-Komplex bildet.
Rafts: Rafts sind spezialisierte Domänen biologischer Membranen, die sich durch ihre spezifische Lipid- und Proteinzusammensetzung auszeichnen (zur Übersicht siehe Simons und Toomre, 2000). Die am besten beschriebenen Rafts sind die Caveolae, doch es gibt noch weitere weniger gut charakterisierte Rafttypen. Rafts werden verschiedene zelluläre Funktionen zugeschrieben wie z.B. gerichteter Transport von Membranproteinen, Endozytose und Signaltransduktion. Diese Funktionen erfüllen sie vornehmlich, indem sie verschiedene Proteine und Lipide bedingt durch ihre biophysikalischen Eigenschaften selektiv aufnehmen oder ausschließen. Viele Raftproteine sind über gesättigte Acylketten, wie Myristat oder Palmitat, oder einen GPIAnker mit der Membran assoziiert. Transmembranproteine, wie z.B. der EGFRezeptor, können jedoch auch in Rafts angereichert sein. Besonders an der Plasmamembran dienen Rafts als Signaltransduktionszentren, indem sie beteiligte Rezeptoren und Signalmoleküle konzentrieren.
Reggie-Proteine: Bei der Suche nach Proteinen, die bei der Regeneration von verletzten Sehnerven von Fischen hochreguliert werden, wurden Reggie-1 und Reggie-2 entdeckt (Schulte et al., 1997). Gleichzeitig wurden diese Proteine bei der Suche nach neuen Raftproteinen gefunden und als Flotillin-1 (=Reggie-2) und Flotillin-2 (=Reggie-1) bezeichnet (Bickel et al., 1997). Reggie-1 und -2 haben ein Molekulargewicht von 47 kDa und sind auf Aminosäuren-Basis zu 44% identisch. Homologe zu Reggie-1 wurden bislang in Mensch, Maus, Ratte und Fisch, wie auch in D. melanogaster gefunden. Die evolutionäre Konservierung der Reggies ist, mit beispielsweise 80% zwischen Ratte und Goldfisch, sehr hoch und weist auf eine wichtige Funktion hin, die Sequenzkonservierung verlangt. Reggie-1 wird ubiquitär exprimiert, wogegen Reggie-2 ein weniger verbreitetes Expressionsmuster aufweist. Reggie-1 ist vornehmlich an der Plasmamembran und an Endosomen lokalisiert. Die subzelluläre Lokalisation von Reggie-2 hängt vom Zelltyp ab...
Keystone mutualisms, such as corals, lichens or mycorrhizae, sustain fundamental ecosystem functions. Range dynamics of these symbioses are, however, inherently difficult to predict because host species may switch between different symbiont partners in different environments, thereby altering the range of the mutualism as a functional unit. Biogeographic models of mutualisms thus have to consider both the ecological amplitudes of various symbiont partners and the abiotic conditions that trigger symbiont replacement. To address this challenge, we here investigate 'symbiont turnover zones'--defined as demarcated regions where symbiont replacement is most likely to occur, as indicated by overlapping abundances of symbiont ecotypes. Mapping the distribution of algal symbionts from two species of lichen-forming fungi along four independent altitudinal gradients, we detected an abrupt and consistent β-diversity turnover suggesting parallel niche partitioning. Modelling contrasting environmental response functions obtained from latitudinal distributions of algal ecotypes consistently predicted a confined altitudinal turnover zone. In all gradients this symbiont turnover zone is characterized by approximately 12°C average annual temperature and approximately 5°C mean temperature of the coldest quarter, marking the transition from Mediterranean to cool temperate bioregions. Integrating the conditions of symbiont turnover into biogeographic models of mutualisms is an important step towards a comprehensive understanding of biodiversity dynamics under ongoing environmental change.
Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death.
In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
Der Hirntumor Glioblastom (GBM) ist aufgrund seines infiltrativen Wachstums, der hohen intra- und intertumoralen Heterogenität, der hohen Therapieresistenz als auch aufgrund der sogenannten gliomartigen Stammzellen sehr schwer zu behandeln und führt fast immer zu Rezidiven. Da es in den letzten Jahrzehnten kaum Fortschritte in der Behandlung des GBMs gab, bis auf die Therapie mit Tumortherapiefeldern, wird weiterhin nach alternativen Zelltodtherapien geforscht, wie zum Beispiel dem Autophagie-abhängigen Zelltod. Der Autophagie-abhängige Zelltod ist durch einen erhöhten autophagischen Flux gekennzeichnet und obwohl die Autophagie, als auch selektive Formen wie die Lysophagie und Mitophagie, normalerweise als überlebensfördernde Mechanismen gelten, konnten viele Studien eine duale Rolle in der Tumorentstehung, -progression und -behandlung aufzeigen, die vor allem vom Tumortyp und stadium abhängt. Um die zugrunde liegenden Mechanismen des durch Medikamente induzierten Autophagie-abhängigen Zelltods im GBM weiter zu entschlüsseln, habe ich in meiner Dissertation verschiedene Substanzen untersucht, die einen Autophagie-abhängigen Zelltod induzieren.
In einer zuvor in unserem Labor durchgeführten Studie konnte gezeigt werden, dass das Antipsychotikum Pimozid (PIMO) und der Opioidrezeptor-Antagonist Loperamid (LOP) einen Autophagie-abhängigen Zelltod in GBM Zellen induzieren können. Darauf aufbauend habe ich die Fähigkeit zur Induktion des Autophagie-abhängigen Zelltods in weiteren Zellmodellen validiert. Dies bestätigte einen erhöhten autophagischen Flux nach PIMO und LOP Behandlung, während der Zelltod als auch der autophagische Flux in Autophagie-defizienten Zellen reduziert war. In weiteren Versuchen konnte ich die Involvierung der LC3-assoziierten Phagozytose (LAP), ein Signalweg der auf die Funktion einiger autophagischer Proteine angewiesen ist, ausschließen. Weiterhin konnte ich eine massive Störung des Cholesterin- und Lipidstoffwechsels beobachten. Unter anderem akkumulierte Cholesterin in den Lysosomen gefolgt von massiven Schäden des lysosomalen Kompartiments und der Permeabiliserung der lysosomalen Membran. Dies trug einerseits zur Aktivierung überlebensfördernder Lysophagie als auch der Zell-schädigenden „Bulk“-Autophagie bei. Letztendlich konnte aber die erhöhte Lysophagie die Zellen nicht vor dem Zelltod retten und die Zellen starben einen Autophagie-abhängigen lysosomalen Zelltod. Da die Eignung von LOP als Therapie für das GBM aufgrund der fehlenden Blut-Hirn-Schranken Permeabilität und von dem Antipsychotikum PIMO aufgrund teils schwerer Nebenwirkungen eingeschränkt ist, habe ich mich im weiteren Verlauf meiner Dissertation mit einer Substanz mit einem anderen Wirkmechanismus beschäftigt.
Der Eisenchelator und oxidative Phosphorylierungs (OXPHOS) Inhibitor VLX600 wurde zuvor berichtet mitochondriale Dysfunktion und Zelltod in Kolonkarzinomzellen zu induzieren. Allerdings hat meines Wissens nach bisher noch keine Studie die therapeutische Eignung von VLX600 für das GBM untersucht. Hier zeige ich eine neuartige Autophagie-abhängige Zelltod-induzierende Fähigkeit von VLX600 für GBM Zellen, da der Zelltod signifikant in Autophagie-defizienten Zellen aber nicht durch Caspase-Inhibitoren gehemmt wurde und der autophagische Flux erhöht war. Darüber hinaus konnte ich die Hemmung der OXPHOS und die Induktion von mitochondrialem Stress in GBM Zellen bestätigen und weiterhin aufzeigen, dass VLX600 nicht nur die mitochondriale Homöostase stört, sondern auch zu einer BNIP3-BNIP3L-abhängigen Mitophagie führt, die wahrscheinlich durch HIF1A reguliert wird aber keinen erkennbaren Nettoeffekt auf den von VLX600 induzierten Zelltod hat. Demnach induziert VLX600 letale „Bulk“-Autophagie in den hier verwendeten Zellmodellen. Darüber hinaus konnte ich zeigen, dass die Eisenchelatierung durch VLX600 eine große Rolle für den von VLX600-induzierten Zelltod spielt aber auch für die Mitophagie Induktion, Histon Lysin Methylierung und den ribosomalen Stress. Letztendlich ist es wahrscheinlich ein Zusammenspiel all dieser Faktoren, die zur Zelltodinduktion durch VLX600 führen und interessanterweise werden Eisenchelatoren bereits in präklinischen und klinischen Studien für Krebstherapien untersucht. Dabei könnten gewisse metabolische Eigenschaften verschiedener Tumorzellen die Sensitivität von Wirkstoffen, die auf den Metabolismus wirken wie VLX600, beeinflussen was in zukünftigen Studien beachtet werden sollte um den bestmöglichsten Therapieerfolg zu erzielen. Zusammenfassend unterstützt meine Dissertation die duale Rolle der Autophagie, die stark vom jeweiligen Kontext abhängt und befürwortet die weitere Forschung von Substanzen, die einen Autophagie-abhängigen Zelltod induzieren, für das GBM.
EphrinB2 and GRIP1 stabilize mushroom spines during denervation-induced homeostatic plasticity
(2021)
Highlights
• Denervation induces mushroom spine loss and AMPAR redistribution to the surface
• GRIP1 and ephrinB2 mediate homeostatic mechanisms after lesion
• Stimulation with the ephrinB2 receptor EphB4 promotes a surface shift of AMPARs
• AMPARs surface shift restores impaired spine recovery after lesion in GRIP1 mutants
Summary
Despite decades of work, much remains elusive about molecular events at the interplay between physiological and structural changes underlying neuronal plasticity. Here, we combined repetitive live imaging and expansion microscopy in organotypic brain slice cultures to quantitatively characterize the dynamic changes of the intracellular versus surface pools of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) across the different dendritic spine types and the shaft during hippocampal homeostatic plasticity. Mechanistically, we identify ephrinB2 and glutamate receptor interacting protein (GRIP) 1 as mediating AMPAR relocation to the mushroom spine surface following lesion-induced denervation. Moreover, stimulation with the ephrinB2 specific receptor EphB4 not only prevents the lesion-induced disappearance of mushroom spines but is also sufficient to shift AMPARs to the surface and rescue spine recovery in a GRIP1 dominant-negative background. Thus, our results unravel a crucial role for ephrinB2 during homeostatic plasticity and identify a potential pharmacological target to improve dendritic spine plasticity upon injury.
Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.
Ecophysiological studies on Antarctic cryptophytes to assess whether climatic changes such as ocean acidification and enhanced stratification affect their growth in Antarctic coastal waters in the future are lacking so far. This is the first study that investigated the combined effects of increasing availability of pCO2 (400 and 1000 µatm) and irradiance (20, 200 and 500 μmol photons m−2 s −1) on growth, elemental composition and photophysiology of the Antarctic cryptophyte Geminigera cryophila. Under ambient pCO2, this species was characterized by a pronounced sensitivity to increasing irradiance with complete growth inhibition at the highest light intensity. Interestingly, when grown under high pCO2 this negative light effect vanished and it reached highest rates of growth and particulate organic carbon production at the highest irradiance compared to the other tested experimental conditions. Our results for G. cryophila reveal beneficial effects of ocean acidification in conjunction with enhanced irradiance on growth and photosynthesis. Hence, cryptophytes such as G. cryophila may be potential winners of climate change, potentially thriving better in more stratified and acidic coastal waters and contributing in higher abundance to future phytoplankton assemblages of coastal Antarctic waters.
Identification of unique cardiolipin and monolysocardiolipin species in Acinetobacter baumannii
(2017)
Acidic glycerophospholipids play an important role in determining the resistance of Gram-negative bacteria to stress conditions and antibiotics. Acinetobacter baumannii, an opportunistic human pathogen which is responsible for an increasing number of nosocomial infections, exhibits broad antibiotic resistances. Here lipids of A. baumannii have been analyzed by combined MALDI-TOF/MS and TLC analyses; in addition GC-MS analyses of fatty acid methyl esters released by methanolysis of membrane phospholipids have been performed. The main glycerophospholipids are phosphatidylethanolamine, phosphatidylglycerol, acyl-phosphatidylglycerol and cardiolipin together with monolysocardiolipin, a lysophospholipid only rarely detected in bacterial membranes. The major acyl chains in the phospholipids are C16:0 and C18:1, plus minor amounts of short chain fatty acids. The structures of the cardiolipin and monolysocardiolipin have been elucidated by post source decay mass spectrometry analysis. A large variety of cardiolipin and monolysocardiolipin species were found in A. baumannii. Similar lysocardiolipin levels were found in the two clinical strains A. baumannii ATCC19606T and AYE whereas in the nonpathogenic strain Acinetobacter baylyi ADP1 lysocardiolipin levels were highly reduced.
In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 – 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content − and thus an interplay of different environmental factors.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
Environmental gradients have emerged as important barriers to structuring populations and species distributions. We set out to test whether the strong salinity gradient from the marine North Sea to the brackish Baltic Sea in northern Europe represents an ecological and genetic break, and to identify life history traits that correlate with the strength of this break. We accumulated mitochondrial cytochrome oxidase subunit 1 sequence data, and data on the distribution, salinity tolerance, and life history for 28 species belonging to the Cnidaria, Crustacea, Echinodermata, Mollusca, Polychaeta, and Gastrotricha. We included seven non-native species covering a broad range of times since introduction, in order to gain insight into the pace of adaptation and differentiation. We calculated measures of genetic diversity and differentiation across the environmental gradient, coalescent times, and migration rates between North and Baltic Sea populations, and analyzed correlations between genetic and life history data. The majority of investigated species is either genetically differentiated and/or adapted to the lower salinity conditions of the Baltic Sea. Species exhibiting population structure have a range of patterns of genetic diversity in comparison with the North Sea, from lower in the Baltic Sea to higher in the Baltic Sea, or equally diverse in North and Baltic Sea. Two of the non-native species showed signs of genetic differentiation, their times since introduction to the Baltic Sea being about 80 and >700 years, respectively. Our results indicate that the transition from North Sea to Baltic Sea represents a genetic and ecological break: The diversity of genetic patterns points toward independent trajectories in the Baltic compared with the North Sea, and ecological differences with regard to salinity tolerance are common. The North Sea–Baltic Sea region provides a unique setting to study evolutionary adaptation during colonization processes at different stages by jointly considering native and non-native species.
Anthropogenic activities have a major impact on our planet and rapidly drive biodiversity loss in ecosystems at a global scale. Particularly over the last century, rising CO2 emissions significantly raised global temperatures and increased the intensity and frequency of droughts and heatwaves. Additionally, agricultural land use and fossil fuel combustion contribute to the continuous release of nitrogen (N) and phosphorus (P) into ecosystems worldwide through extensive fertilization and deposition from the atmosphere. It is important to understand how these rapid changes affect the evolution of plant populations and their adaptive potential. Adaptation by natural selection (i.e., adaptive evolution) within a few generations is an essential process as a response to rapid environmental changes. Rapid evolution of plant populations can be detected by using the so-called resurrection approach. Here, diaspores (i.e., seeds) from a population are collected before (ancestors) and after (descendants) a potential selection pressure (e.g., consecutive years of drought or changes in nutrient supply). Comparing phenotypes of ancestors and descendants in a common environment such as an outside garden, greenhouse, or climate chamber, may then reveal evolutionary changes. Ideally, plants are first grown in a common environment for an intermediate refresher generation to reduce parental and storage effects.
The aim of this thesis was to investigate the occurrence of adaptive evolution in natural plant populations in response to rapidly changing environments over the past three decades. I conducted three experiments using the resurrection approach to generate comprehensive data on the adaptive processes that acted on three plant populations from three different species over the last three decades. Furthermore, I filled knowledge gaps in plant evolutionary ecology and conceptually developed the resurrection approach further.
In Chapter I, I performed a novel approach by testing for adaptive evolution in natural plant populations using the resurrection approach in combination with in-situ transplantations. I cultivated seedlings from ancestors (23 – 26 years old) and contemporary descendants of three perennial species (Melica ciliata, Leontodon hispidus and Clinopodium vulgare) from calcareous grasslands in the greenhouse and In Chapter III, I assessed the reproducibility of phenotypic differences between genotypes among three different growth facilities (climate chamber, greenhouse, and outdoor garden). I also evaluated differences in phenotypic expression between plants grown after one vs. two intermediate generations (i.e., refresher generations). I performed this experiment within the framework of the resurrection approach and compared ancestors and descendants of the same population of Leontodon hispidus.
I observed very strong differences among plants growing in the different growth facilities. I found a significant interaction between the growth facility and the temporal origin (ancestors vs. descendants): descendants had significantly larger rosettes than ancestors only in the greenhouse and they flowered significantly later than ancestors exclusively in the climate chamber. I did not find significant differences between intermediate generations within the growth facilities. Overall, Chapter III shows that the use of a particular experimental system can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence is not evidence of absence when it comes to investigating genetically based trait differentiation among plant origins (in space or time). Experimental systems should be carefully designed to provide meaningful conditions, ideally mimicking the environmental conditions of the population’s origins. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the environments, supporting the idea that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
The resurrection approach allows a better understanding of rapid plant adaptation, but some limitations deserve to be highlighted. I only studied one population per species, and Chapters II and III only focus on one population of L. hispidus, which is also hampering generalizations, as adaptive potential can vary greatly among populations of the same species. I only compared the ancestral genotypes to one descendant sample with a long time span in between (26 – 28 years), which makes it hard to pinpoint the selection agents that caused the genetic differentiation among the sampling years. Hence, closely monitoring biotic and abiotic factors of the studied populations between the ancestral and descendant sampling in future studies, would make identifying the responsible selection pressures more precise. I also recommend sampling multiple populations over consecutive years to improve the robustness of results and make generalizations more approachable.Furthermore, combining the resurrection approach with other methods such as in-situ transplantations will be valuable to offset the limitation that adaptations cannot be proven under artificial conditions (e.g., in the greenhouse).
In Arabidopsis thaliana, the stem cell niche (SCN) within the root apical meristem (RAM) is maintained by an intricate regulatory network that ensures optimal growth and high developmental plasticity. Yet, many aspects of this regulatory network of stem cell quiescence and replenishment are still not fully understood. Here, we investigate the interplay of the key transcription factors (TFs) BRASSINOSTEROID AT VASCULAR AND ORGANIZING CENTRE (BRAVO), PLETHORA 3 (PLT3) and WUSCHEL-RELATED HOMEOBOX 5 (WOX5) involved in SCN maintenance. Phenotypical analysis of mutants involving these TFs uncover their combinatorial regulation of cell fates and divisions in the SCN. Moreover, interaction studies employing fluorescence resonance energy transfer fluorescence lifetime imaging microscopy (FRET-FLIM) in combination with novel analysis methods, allowed us to quantify protein-protein interaction (PPI) affinities as well as higher-order complex formation of these TFs. We integrated our experimental results into a computational model, suggesting that cell type specific profiles of protein complexes and characteristic complex formation, that is also dependent on prion-like domains in PLT3, contribute to the intricate regulation of the SCN. We propose that these unique protein complex ‘signatures’ could serve as a read-out for cell specificity thereby adding another layer to the sophisticated regulatory network that balances stem cell maintenance and replenishment in the Arabidopsis root.
Im Rahmen der vorliegenden Arbeit werden zum ersten Mal die Ökologie, Morphologie und Systematik von Pilzen untersucht, die assoziiert mit Haut- und Nagelläsionen von ambulanten Patienten sowie von Patienten dermatologischer Praxen in der Provinz Chiriquí im Westen Panamas nachgewiesen wurden. Die Pilze werden klassifiziert nach dem klinischen D-H-SSystem von Rieth und entsprechend ihrer Position im phylogenetischen System der Pilze. Die Morphologie der verschiedenen Arten wird dokumentiert auf der Grundlage von Kulturen und lichtmikroskopischer Untersuchungen durch Beschreibungen sowie Zeichnungen und Fotographien charakteristischer Strukturen. Die Pathogenität der einzelnen Pilzstämme wurde nicht nachgewiesen, sondern auf der Grundlage von Angaben aus der Literatur diskutiert. Außerdem lieferte die Literatur Daten zum Vorkommen der Pilze an Pflanzen und anderen Substraten in der Natur.
In Panama wurden zahlreiche klinische Proben untersucht, von denen ca. 100 Pilzstämme nach Deutschland geschickt wurden. Dort konnten 80 Stämme weiter kultiviert und detailliert untersucht werden. Mehr als 22 verschiedene Arten wurden beobachtet, die 17 verschiedenen Gattungen angehören. Sie entsprechen drei verschiedenen Arten von Dermatophyten, mindestens drei Arten von Hefen und 16 verschiedenen Schimmel- oder sonstigen Pilzarten.
Mit Ausnahme von Hormographiella verticillata wurden ausschließlich imperfekte Stadien beobachtet, und zwar überwiegend von verschiedenen Vertretern der Ascomycota: Dothideales: Scytalidium dimidiatum (6 Stämme), Eurotiales: Aspergillus spp. (4), Paecilomyces lilacinus (2), Penicillium sp. (2), Hypocreales: Fusarium lichenicola (3), F. solani (4), F. subglutinans (1), Microascales: Scopulariopsis brevicaulis (2), Onygenales: Trichophyton mentagrophytes (2), T. rubrum (9), T. tonsurans (7), Ophiostomatales: Sporothrix schenckii (1), Pleosporales: Curvularia geniculata (1), Polystigmatales: Colletotrichum gloeosporioides (1), Sordariales: Nigrospora sphaerica (1), Saccharomycetales: Candida spp. (12), Geotrichum candidum (8), incerte sedis: Pestalotiopsis cf. tecomicola (1), Tritirachium oryzae (1). Vertreter der Basidiomycota sind: Agaricales: Hormographiella verticillata bzw. Coprinellus domesticus (3), Polyporales: Unbekannter Basidiomycet (1), Trichosporonales: Trichosporon cutaneum (6).
Im Rahmen dieser Studie waren Schimmelpilze die am häufigsten bei Haut- und Nagelläsionen angetroffenen Pilze. Unter diesen waren Fusarium-Arten und Scytalidium dimidiatum besonders häufig vertreten. Candida-Arten wurden ebenfalls oft isoliert. Die wichtigste Art unter den Dermatophyten war Trichophyton rubrum. Die prozentualen Anteile der verschiedenen Gruppen entsprechen gut den von anderen Autoren aus anderen Regionen publizierten Ergebnissen. Dies erklärt sich aufgrund der ökologischen Tatsache, dass die Sporen der Schimmelpilze fast überall in der Natur vorhanden sind und diese Pilze viele verschiedene Substrate nutzen können. Candida-Arten gehören zur normalen Flora des Menschen, können aber bei immunodefizienten Patienten, Diabetikern u.a. schwere Haut- und Schleimhautinfektionen, sowie Organerkrankungen verursachen. Dermatophyten sind als Krankheitserreger oberflächlicher Hautmykosen bekannt.
Zum ersten Mal wird das Vorkommen von Hormographiella verticillata in Amerika nachgewiesen. Dieses imperfekte Stadium eines Basidiomyceten hat in Kultur Fruchtkörper gebildet, die als Coprinellus domesticus bestimmt wurden. Damit wurde zum ersten Mal die Anamorph-Teleomorph-Verbindung zwischen diesen beiden Arten festgestellt, die durch eine molekular-phylogenetische Analyse von LSU rDNA (große Untereinheit der ribosomalen DNA) unterstützt wird. Für diese Analyse wurden andere Stämme und Genbank-Daten zum Vergleich herangezogen.
In den Kulturen von H. verticillata entstehen vor der Entwicklung der Fruchtkörper asexuelle sterile Hyphen, die als Ozonium-Stadium bezeichnet werden können. Zum Vergleich wurden Herbarbelege von verschiedenen Arten dieser Gattung bearbeitet. Die Arten sind morphologisch nicht unterscheidbar, weshalb vorgeschlagen wird, nur den Gattungsnamen zur Bezeichnung des entsprechenden Entwicklungsstadiums zu benutzen.
Es war nicht möglich, aufgrund morphologischer Merkmale den Stamm des Unbekannten Basidiomyceten zu bestimmen. Erst eine molekular-phylogenetischer Analyse von LSU rDNA mit Vergleichssequenzen aus der Genbank zeigte, dass der Pilz nahe verwandt ist mit Vertretern der Polyporales.
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5′-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
Alzheimer’s disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aβ) in the brain, which leads to progressive cognitive deficits and dementia. Aβ is generated by the successive cleavage of the amyloid precursor protein (APP), first by β-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aβ aggregation and the development of AD. In vitro studies have demonstrated that Aβ assemblies spark a feed-forward loop heightening Aβ production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aβ enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aβ42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aβ or to activate Go protein, we have found that treatment with aggregated Aβ fails to increase colocalization of APP with BACE1 indicating that Aβ-APP/Go signaling is involved in this process. Moreover, inhibition of Gβγ subunit signaling with βARKct or gallein prevents Aβ-dependent interaction of APP and BACE1 in endosomes, β-processing of APP, and intracellular accumulation of Aβ42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aβ pathology in the early stages of AD and suggest that gallein could have therapeutic potential.
In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host’s anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens’ characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased (“processing escapes”). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
Das positions-spezifisch integrierende TRE5-A Retrotransposon besitzt zwei Promotorregionen (A- und C-Modul). Für das C-Modul konnte ein spezifisch bindendes Protein (CbfA) gefunden werden, das vermutlich über die Expression des Minusstrang-Transkripts regulativ in den Transpositionsmechanismus eingreift. Gleichzeitig stellt CbfA in D. discoideum einen kritischen Faktor dar, der sowohl auf das Wachstum als auch auf die Differenzierung Einfluss nimmt.
Es konnte gezeigt werden:
• Der AT-Haken in CbfA ist für die DNA-Bindung essentiell. Die Inaktivierung hat zur Folge, dass keine Differenzierung stattfindet. Es scheint, dass primär der AT-Haken für die DNA-Bindung sorgt und von den Zinkfingern unterstützt wird.
• Die JmjC-Domäne in CbfA ist essentiell. Transformanden mit CbfA ohne aktive JmjC-Domäne zeigen Defekte sowohl in Wachstum als auch Differenzierung.
• CbfA ist ein nukleares Protein. Es konnte zwar keine Kernlokalisationssequenz identifiziert werden, jedoch weisen die Versuche auf zumindest eine Kernlokalisierungssequenz im C-Terminus des cbfA hin.
• Ein C-terminal verkürztes CbfA ist funktionslos: wahrscheinlich aufgrund fehlender Kernlokalisation und somit fehlender DNA-Bindung.
• Ein N-terminal verkürztes CbfA ist teilweise funktionsfähig: die Komplementation in JH.D2-Zellen führt zu einer partiellen Revertierung hin zum Phänotyp der AX2-Zellen.
• Struktur-Homologien der JmjC-Domäne in CbfA zu Mitgliedern aus der Familie der Fe(II)/2OG-Oxygenasen, DNA-bindende Motive und die Lokalisation im Zellkern weisen auf eine Funktion des CbfAs als Chromatin-Remodeller im Zellkern hin.
• In der Transit von Wachstums- zu Entwicklungsphase kann der CbfA-Mangel durch artifizielle Proteinase A-Expression ausgeglichen werden, aber nicht durch Komplementation mit YakA, Adenylat-Zyklase oder cAMP Rezeptor 1.
• CbfA fungiert wahrscheinlich als Regulator der acaA-Transkription auf Ebene der chromosomalen Strukturen.
• cbfB, ein weiteres Gen mit einer JmjC-Domäne wurde in D. discoideum identifiziert, die Gensequenz vervollständigt und vom Dictyostelium Genom-Projekt verifiziert.
Diadenosinpolyphosphate (ApnAs), wirken in einer Vielzahl unterschiedlicher Gewebe als extrazelluläre Signalmoleküle. Ihre asymmetrische Hydrolyse durch Enzyme auf der Zelloberfläche oder durch lösliche Enzyme im extrazellulären Milieu wurde in der Literatur bereits mehrfach beschrieben. Die molekulare Identität dieser Enzyme war jedoch nicht bekannt.
In der vorliegenden Arbeit wurden die Fähigkeit von NPP1, NPP2 und NPP3, den drei Mitgliedern der E-NPP-Familie, Diadenosinpolyphosphate zu hydrolysieren, untersucht. Dazu wurden humanes NPP1 und NPP3 aus der Ratte heterolog in CHO-Zellen exprimiert und die enzymatische Aktivität wurde anhand von Membranfraktionen analysiert. Für die Charakterisierung der katalytischen Eigenschaften von NPP2 wurde eine lösliche sekretierte Form des humanen NPP2 aus partiell aufgereinigtem Vaccinia-Viruslysat eingesetzt. Es konnte gezeigt werden, dass die heterolog exprimierten Enzyme NPP1, NPP2 und NPP3 die untersuchten Diadenosinpolyphosphate Diadenosin-5´,5´´´-P1,P3-triphosphat (Ap3A), Diadenosin-5´,5´´´-P1,P4-tetraphosphat (Ap4A) und Diadenosin-5´,5´´´-P1,P5-pentaphosphat (Ap5A), sowie das Diguanosinpolyphosphat Diguanosin-5´,5´´´-P1,P4-tetraphosphat (Gp4G) hydrolysieren.. Ein Vergleich der Hydrolyseraten zeigte, dass NPP1-2 Ap3A, Ap4A, Ap5A und Gp4G mit vergleichbarer Rate hydrolysieren. NPP3 zeigte ebenfalls keine Präferenz für eines der untersuchten Diadenosinpolyphosphate, hydrolysierte aber Gp4G im Vergleich zu Ap4A deutlich langsamer. Die Hydrolyse der Dinukleotide erfolgte asymmetrisch durch Spaltung der α,β-Pyrophosphatbindung. Als primäre Hydrolyseprodukte entstanden Nukleosid-5´-Monophosphat und der verbleibende Mononukleotidrest Npn-1. Für Ap3A als Substrat wurde für NPP1-3 ein alkalisches pH-Optimum mit maximaler Aktivität bei pH 8.5-9 (NPP1 und NPP3), bzw. pH 10 (NPP2) nachgewiesen. Die enzymatische Aktivität von NPP1-3 wurde durch EDTA inhibiert und die Km-Werte für Ap3A lagen mit 5,1 ± 3,6 µM (NPP1), 8,0 ± 0,5 µM (NPP2) und 49,5 ± 17,7 µM (NPP3) im niedrigen mikromolaren Bereich. Untersuchungen zur Hemmbarkeit der NPP-vermittelten Diadenosinpolyphosphathydrolyse (Ap4A) zeigten, dass die Enzyme NPP1, NPP2 und NPP3 nach Koapplikation verschiedener P2-Rezeptorantagonisten in unterschiedlichem Ausmaß inhibiert wurden. Cibacron Blue inhibierte kräftig alle drei Enzyme, während PPADS einen stärkeren inhibitorischen Einfluss auf die katalytsiche Aktivität von NPP1 und NPP3 als auf die katalytische Aktivität von NPP2 zeigte. Suramin inhibierte dagegen nur die NPP1 und NPP2 katalysierte Ap4AHydrolyse und hatte keinen Einfluss auf NPP3. Eine Inhibierung von NPP1-3 durch NaF wurde nicht beobachtet. Eine geringe inhibitorische Wirkung auf NPP1 wurde durch die extrazellulären Matrix-Komponenten Heparin und Heparansulfat, durch ATP und die Nukleotidanaloga, AMP-CP, AMP-CPP und ATP-γ-S beobachtet. NPP2 und NPP3 wurden durch AMP-CP nicht inhibiert. Den schwächsten inhibitorischen Einfluss zeigte ATP auf die durch NPP3-vermittelte Ap4AHydrolyse.
Die hier für NPP1-3 ermittelten katalytischen Eigenschaften zeigten Übereinstimmgen aber auch Unterschiede gegenüber früheren Daten, die für die Hydrolyse von Diadenosinpolyphosphaten auf der Oberfläche von Zellen ermittelt wurden. Insgesamt sprechen die Ergebnisse dafür, dass NPP1-3 die Hauptvertreter der Diadenosinpolyphosphat-hydrolysierenden Enzyme in Säugergewebe darstellen. Möglicherweise gibt es aber auch ApnA-hydrolysierende Enzyme, die nicht zu den bisher charakterisierten Mitgliedern der E-NPP-Familie zugehören.
In einem zweiten Teil der Arbeit wurde die Expression von NPP1-3 im Gehirn der Ratte mittels Western-Blot-Analyse (Entwicklungsstadien P1, P21 und adult) untersucht. Aufgrund der geringen Spezifität der gegen NPP1 und NPP2 zur Verfügung stehenden Antikörper, konnten jedoch keine eindeutigen Aussagen zur Expression von NPP1 und NPP2 im Gehirn getroffen werden. NPP3 konnte im Rattengehirn nachgewiesen werden. Die Expression war entwicklungsabhängig und nahm mit zunehmendem Alter der Tiere deutlich ab. Die Entwicklung spezifischer Antikörper erscheint ein lohnender Ansatz, um die zelluläre Verteilung von NPP1-3 im Nervengewebe zu bestimmen.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
Spaceflight affects the body on every level. Reports on astronaut health identify bone marrow remodelling and dysfunction of the innate immune system as significant health risks of long-term habitation in space. Microgravity-induced alterations of the bone marrow induce physical changes to the bone marrow stem cell niche. Downstream effects on innate immunity are expected due to impaired hematopoiesis and myelopoiesis. To date, few studies have investigated these effects in real microgravity and the sparsely available literature often reports contrasting results. This emphasizes a need for the development of physiologically relevant in vitro models of the bone marrow stem cell niche, capable of delivering appropriate sample sizes for robust statistics. Here, we review recent findings on the impact of spaceflight conditions on innate immunity in in vitro and animal models and discusses the latest in vitro models of the bone marrow stem cell niche and their potential translatability to gravitational biology research.
Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
Fungi belonging to the Rhytismatales (Ascomycota) are parasites or endophytes of plants, some are saprophytes. Their fruiting bodies are localized in different organs of the host plants belonging to many different families of gymnosperms and angiosperms. Many species of Rhytismatales are known on species of Pinaceae, Ericaceae, and Poaceae. These fungi usually have ascomata that are more or less embedded in host tissue and open by longitudinal or radial splits. They have a more or less carbonized covering stroma, thin-walled, iodine negative asci, and ascospores usually covered by gelatinous sheaths.
In the present study, two lists of species of Rhytismatales in China are presented. One is based on literature and includes 103 species in 15 genera. The second one contains the names of the species in the present study, 57 species in 20 genera based on 90 specimens I collected in the Yunnan and Anhui province in China during July to August in 2001. 31 species in the second list are new species or new records for China, so we presently know 134 species in 22 genera of Rhytismatales for China. 28 new species of Rhytismatales are proposed, 21 species from the Yunnan province and seven from the Anhui province. Among them, three new species are proposed in three new genera, Nematococcomyces, New Genus 1, and New Genus 2, respectively. The 28 new species are Cerion sp., Coccomyces spp. 1-2, Colpoma spp. 1-2, Hypoderma spp. 1-6, Lirula sp., Lophodermella sp., Lophodermium spp. 1-5, Nematococcomyces rhododendri C.-L. Hou, M. Piepenbr. & Oberw., Neococcomyces sp., New Genus 1 sp., New Genus 2 sp., Rhytisma spp. 1-2, Soleella sp., Terriera spp. 1-2, and Therrya sp. The genus Davisomycella is proposed as a synonym of Lophodermella based on observations of the morphology, ecology, and the infected organ. The four genera Cerion, Naemacyclus, Terriera, and Therrya, and three species, Hypoderma rubi, Lophodermium uncinatum, and Naemacyclus pinastri, are reported for the first time for China. All the new taxa, the newly recorded ones, as well as six species which had not been illustrated in detail before, are carefully described and illustrated by line drawings in the present study.
The results show that species of Rhytismatales are highly diverse especially in the natural vegetation in high mountainous areas in China. Most species of Rhytismatales are conspicuously host specific. The diversity of Rhytismatales is closely related to that of the preferred hosts, which are members of Pinaceae, Ericaceae, and Cupressaceae. Based on the detailed morphological observations, the significance of different morphological characteristics for a natural classification of Rhytismatales is discussed. Genera are traditionally defined by character states of a few characteristics, namely the opening patterns of ascomata, the depth of ascomata in the host tissue, and asci and ascospore shape. Data from collections in the field, detailed morphological investigation, and molecular data show, however, that the ecology, the infected organ, the host relationship, and many other characteristics have to be combined to circumscribe natural groups.
The discussion of the systematic significance of morphological characteristics is complemented by molecular data. In the present study, partial nuclear large subunit rDNA sequences of 52 specimens representing 38 species are used to analyse phylogenetic relationships for members of Rhytismatales.
Most species of Rhytismatales are placed in a monophyletic group corresponding to the Rhytismatales in the Maximum Parsimony analysis. The delimitation of the Rhytismatales from the Helotiales is, however, difficult. Cyclaneusma minus should be transferred from the Rhytismatales to the Helotiales, and Cudonia circinans and Spathularia flavida from the Helotiales to the Rhytismatales. These tranfers have previously been proposed based on SSU rDNA analysis by other authors. New Genus 1 sp. has morphological characteristics typical for species of Rhytismatales. In the LSU rDNA analysis, however, it is more closely related to Helotiales rather than toRhytismatales. Therefore New Genus 1 sp. is placed in the Helotiales.
Tryblidiopsis pinastri is morphologically intermediate between members of Rhytismataceae and Cudoniaceae. LSU rDNA sequences in the present study show that T. pinastri is more closely related to species of Cudoniaceae. Therefore, this species is removed from the Rhytismataceae to the Cudoniaceae. The delimitation of further families could not be resolved in the present analysis.
Though many new morphological, ecological, and molecular phylogenetic findings are contributed for the first time, the systematic conclusions at generic, family, and order level can only be fragmentary in the present study. With more collections and more molecular data of the worldwide 450 known and many more unknown species of Rhytismatales at hand, a natural system combining morphological and molecular analysis can be elaborated.
Einleitung
APP und die Alzheimersche Krankheit
Das Alzheimer Amyloid Precursor Protein (APP) ist ein Typ-1 Transmembranprotein mit einem Molekulargewicht von 110-135 kDa [Selkoe et al. 1988, Weidemann et al. 1989]. Es wird in allen bisher untersuchten Geweben exprimiert und weist in mehrzelligen Organismen einen hohen Konservierungsgrad auf [Robakis et al. 1987, Rosen et al. 1989]. APP ist unter anderem Vorläufer des β-A4-Peptides (Aβ), das in extrazellulären Aggregaten (Plaques) im Zentralen Nervensystem von Alzheimer-Patienten akkumuliert [Masters et al. 1985]. Die sogenannte „Amyloid-Hypothese der Alzheimerschen Erkrankung“ besagt, dass das Aβ-Peptid eine pathologische Kaskade initiiert, die zur Bildung von amyloiden Plaques, neuronaler Funktionsstörung und letztendlich Demenz führt [Hardy 1997, Selkoe 1999].
Prozessierung des APP
Der Hauptanteil des zellulären APP wird über den (nicht pathogenen) α-Sekretase-Weg prozessiert, wobei das sekretorische APP (α-sAPP) freigesetzt wird, das beinahe der gesamten N-terminalen Ektodomäne des APP entspricht. Die α-Sekretase spaltet APP innerhalb der Aβ-Domäne und verhindert somit die Bildung des pathogenen Aβ-Peptides. Kandidaten für die Katalyse dieser Spaltung sind Proteasen der ADAM-Familie [Buxbaum et al. 1998, Hooper et al. 1997, Koike et al. 1999, Lammich et al. 1999, Loechel et al. 1998].
Das Aβ-Peptid entsteht bei der sukzessiven proteolytischen Spaltung des APP durch die sogenannten β- und γ-Sekretasen. Bei der β-Sekretase handelt es sich um die Aspartat-Protease BACE (β-site APP cleaving enzyme) [Hussain et al. 1999, Sinha et al. 1999, Vassar et al. 1999, Yan et al. 1999]. Die Identität der γ-Sekretase ist noch nicht endgültig geklärt, jedoch spielen Presenilin-1 und -2 sowie Nicastrin eine Rolle bei der γ-Spaltung des APP [de Strooper et al. 1998, 1999, Struhl et al. 2000, Wolfe et al. 1999].
Unter physiologischen Bedingungen wird ca. 30% des APP durch α-Sekretasen prozessiert, ein viel geringerer Anteil dagegen durch die β-Sekretasen. Mehr als die Hälfte des zellulären APP bleibt ungespalten [Koo 2002].
Biologische Funktionen des APP
Die Funktionen des APP lassen sich unterscheiden nach Funktionen der kurzen zytoplasmatischen Domäne und der ca. 100 kDa großen Ektodomäne (α-sAPP). Die zytoplasmatische Domäne des APP stellt eine Plattform für die Bindung verschiedener Interaktionspartner dar. In Kooperation mit den Bindungspartnern spielt APP eine Rolle in unterschiedlichsten zellulären Prozessen wie vesikulärem Transport, Zellmotilität oder Genaktivierung [Review siehe Annaert und de Strooper 2002]. Die meisten Interaktionspartner der zytoplasmatischen Domäne des APP binden an die YENPTY-Sequenz nahe des C-Terminus des APP, die auch als Signal für die Endozytose des APP dient [Perez et al. 1999].
Die sekretorische Ektodomäne des APP hat eine wachstumsfördernde und neuroprotektive Wirkung. Um diese Wirkung auszuüben, bindet α-sAPP an einen bisher unbekannten Rezeptor, der auf der Zelloberfläche diverser Zelltypen wie Neuronen, Fibroblasten, Thyreozyten und Keratinozyten exprimiert wird [Review siehe Schmitz et al. 2002].
Polarer Transport des APP
In polaren MDCK Zellen wird das APP-Holoprotein fast ausschließlich zur basolateralen Zelloberfläche transportiert [Haass et al. 1994]. Es wurde gezeigt, dass dieser polare Transport des APP durch Tyrosin 653 in der zytoplasmatischen Domäne des APP beeinflusst wird. Mutation dieses Tyrosins zu Alanin führte zu partieller Fehlsortierung von ca. 50% des APP zur apikalen Plasmamembran. Die Sekretion von α-sAPP dagegen fand in MDCK-Zellen unabhängig von Tyrosin 653 basolateral statt [Haass et al. 1995].
Intrazellulärer Proteintransport durch Adaptor-Protein-Komplexe
Am intrazellulären Proteintransport sind Adaptor-Protein-Komplexe (APs) beteiligt, die bestimmte Sortierungssignale in der zytoplasmatischen Domäne von Frachtproteinen erkennen. Bis heute sind vier dieser tetrameren AP-Komplexe (AP-1 bis AP-4) bekannt, die zum Teil verschiedene Isoformen einzelner Untereinheiten aufweisen, z.B. AP-1A und AP-1B [Review: Boehm und Bonifacino 2001]. Jeder AP-Komplex spielt eine Rolle in einem bestimmten Schritt des intrazellulären Proteintransportes. Für AP-1A wird eine Funktion im anterograden und retrograden Transport zwischen Endosomen und TGN beschrieben [Review: Hinners und Tooze 2003]. AP-2 vermittelt Endozytose verschiedener Transmembranproteine von der Plasmamembran [Review: Kirchhausen 2002]. AP-3 spielt eine Rolle im Proteintransport zu Lysosomen und Lysosom-ähnlichen Organellen wie Melanosomen [Robinson und Bonifacino 2001]. AP-4 sowie AP1-B sortieren Proteine zur basolateralen Plasmamembran polarer Epithelzellen [Fölsch et al. 1999, Simmen etal. 2002].
Die Sortierungsmotive, die von Adaptor-Komplexen in der zytoplasmatischen Domäne der Fracht-Proteine gebunden werden, enthalten in den meisten Fällen entweder ein Tyrosin oder zwei Leucine. Das gesamte Motiv besteht aus jeweils vier bis zehn Aminosäuren [Review siehe Bonifacino und Traub 2003].
Ziele der Arbeit
In der vorliegenden Arbeit wurde der polare Transport des APP in Epithelzellen untersucht. Ein Ziel war es, Faktoren zu finden, die den basolateralen Transport des APP in Abhängigkeit von Tyrosin 653 vermitteln. Des weiteren sollte der Transport von APP und sAPP in verschiedenen Epithelzelllinien analysiert werden. Um ein gutes Werkzeug zur Detektion von APP zu haben, wurden GFP-APP-Fusionsproteine hergestellt und charakterisiert.
Ergebnisse und Diskussion
GFP-APP-Fusionsproteine wurden hergestellt und in MDCK-, FRT- und LLC-PK1-Zellen stabil exprimiert. Die Charakterisierung der GFP-APP-Fusionsproteine durch Immunfluoreszenzanalysen zeigte, dass die chimeren Proteine im TGN sowie in peripheren Vesikeln lokalisiert sind und mit endogenem APP stark kolokalisieren. GFPAPP war somit gut geeignet, um den intrazellulären Transport des APP zu untersuchen.
Eine Analyse der zytoplasmatischen Domäne des APP im Bereich des Tyrosin 653 zeigte, dass dieses Tyrosin und die drei folgenden Aminosäuren (YTSI) ein Konsensus-Motiv für die Bindung von tetrameren Adaptor-Protein-Komplexen darstellen.
Zu Beginn dieser Arbeit waren AP-1 bis AP-3 bereits gut charakterisiert, wohingegen für AP-4 keine Funktion bekannt war. In Kollaboration mit Simmen et al. konnte gezeigt werden, dass AP-4 den basolateralen Transport einiger Proteine vermittelt [Simmen et al. 2002]. Immunfluoreszenzanalysen lokalisierten AP-4 im TGN und peripheren Vesikeln, die unterschiedlich von AP-1A/B markierten Strukturen waren. Da kaum Kolokalisation von AP-4 und AP-1A/B zu beobachten war, ist die Lokalisation von AP-4 und AP-1B, das auch eine Rolle im basolateralen Proteintransport spielt, in unterschiedlichen Subdomänen des TGN und unterschiedlichen vesikulären Strukturen anzunehmen.
Polarer Transport des APP durch Adaptor-Protein-Komplexe
Die mögliche Funktion von AP-1 und AP-4 im Transport von APP wurde zunächst mit Hilfe von in vitro-Bindungsstudien untersucht. Dazu wurde die zytoplasmatische Domäne des APP als GST-Fusionsprotein kloniert und exprimiert. Die Frachtproteinbindenden Untereinheiten von AP-1 und AP-4 wurden unter Verwendung von radioaktiv markiertem Methionin durch in vitro-Transkription und -Translation hergestellt. In Bindungsstudien interagierten AP-1A und AP-1B mit der zytoplasmatischen Domäne des APP, nicht aber AP-4. Diese Ergebnisse deuten an, dass AP-1A und AP-1B eine Rolle im intrazellulären Transport von APP spielen könnten. AP-4 dagegen scheint nicht an diesem Prozess beteiligt zu sein.
Durch Mutation des Tyrosin 653 in APP zu Alanin (Y653A) wurde die Interaktion zwischen AP-1B und APP stark verringert, was darauf hindeutet, dass dieses Tyrosin einen Teil des Bindungsmotivs für AP-1B darstellt. Übereinstimmend damit entspricht die genaue Aminosäureabfolge des Y653TSI-Motivs den Sotierungsmotiv-Präferenzen von AP-1B [Ohno et al. 1999]. Die Interaktion von AP-1A dagegen war mit WildtypAPP und der Tyrosin-Mutante vergleichbar und scheint somit auf einem anderen Interaktions-Motiv zu basieren. AP-1A und AP-1B erkennen somit unterschiedliche Sortierungsmotive in der zytoplasmatischen Domäne des APP und kooperieren möglicherweise im intrazellulären Transport des APP. Diese Ergebnisse sind der erste Bericht über eine Interaktion von Adaptor-Protein-Komplexen mit der zytoplasmatischen Domäne des APP.
Die Rolle von AP-1B im basolateralen Transport von APP wurde genauer untersucht mit Hilfe der LLC-PK1 Zelllinie, die kein AP-1B exprimiert [Ohno et al. 1999]. In LLCPK1-Zellen werden verschiedene Proteine unpolar zur apikalen und basolateralen Membran verteilt, die in MDCK-Zellen durch Interaktion mit AP-1B basolateral transportiert werden [Fölsch et al. 1999, Sugimoto et al. 2002]. Um den Transport von APP in polaren LLC-PK1-Zellen zu untersuchen, wurde Plasmamembran-ständiges GFP-APP durch zwei unabhängige Methoden nachgewiesen: die apikale oder basolaterale Oberfläche der Zellen wurde selektiv entweder biotinyliert oder mit GFPAntikörpern markiert. Beide Methoden zeigten, dass GFP-APP in LLC-PK1-Zellen sowohl an der apikalen als auch an der basolateralen Zelloberfläche lokalisiert ist. Somit wird auch APP in diesen Zellen im Vergleich zu MDCK-Zellen anders sortiert. Dieses Ergebnis festigt die Hypothese einer Funktion von AP-1B im Transport von APP, die aufgrund der Daten der in vitro-Bindungsstudien aufgestellt wurde.
Polare Sekretion des sAPP ist unabhängig vom Transport des Holoproteins
Neben dem Transport des APP-Holoproteins war auch die polare Sekretion des sAPP Thema dieser Arbeit. Es war gezeigt worden, dass basolaterale Sekretion des sAPP in MDCK-Zellen unabhängig vom Transport des APP-Holoproteins ist [Haass et al. 1995]. Dieses Ergebnis konnte in der vorliegenden Arbeit bestätigt und auf andere Zelllinien erweitert werden. Um die korrekte Sekretion von GFP-sAPP nachzuweisen, wurde die GFP-sAPP-Sekretion zunächst in polaren MDCK-Zellen untersucht, die stabil GFP-APP exprimierten. Da GFP am N-Terminus des APP angefügt ist, trägt auch das sezernierte APP die GFP-Markierung. GFP-sAPP konnte mittels Immunpräzipitation mit GFP-spezifischen Antikörpern lediglich im basolateralen Medium nachgewiesen werden. Somit sezernieren MDCK-Zellen GFP-sAPP in gleicher Polarität wie von Haass et al. für endogenes sAPP gezeigt wurde [Haass et al. 1995].
Experimente in GFP-APP exprimierenden LLC-PK1- und FRT-Zellen zeigten, dass auch hier die polare Sekretion des GFP-sAPP und der Transport des APPHoloproteins zwei unabhängige Prozesse sind. Polare LLC-PK1-Zellen transportierten GFP-APP zur apikalen und basolateralen Plasmamembran (siehe oben). GFP-sAPP-Sekretion aus polaren LLC-PK1-Zellen dagegen fand ausschließlich basolateral statt. In FRT-Zellen wurde GFP-sAPP im Gegensatz zu MDCK- und LLCPK1-Zellen apikal sezerniert. Kolokalisation des GFP-APP mit Transferrin-Rezeptor in FRT-Zellen deutete dagegen an, dass das Holoprotein wie in MDCK-Zellen basolateral transportiert wird. Dies ist auch zu erwarten, da FRT-Zellen AP-1B exprimieren und es auch in dieser Zelllinie basolateralen Transport vermittelt [A. Gonzalez, persönlich, ASCB 2003]. Nach diesen Ergebnissen zu urteilen, finden auch in FRT und LLC-PK1-Zellen APP-Transport und sAPP-Sekretion unabhängig voneinander statt.
Basolaterale sAPP-Sekretion ist unabhängig von der Ektodomäne
In MDCK-Zellen wurde zusätzlich die Sekretion eines GFP-APP untersucht, in dem der Großteil der Ektodomäne deletiert und durch GFP ersetzt wurde, die SekretaseSchnittstellen jedoch noch vorhanden waren. Durch Immunfluoreszenzanalyse wurde zunächst nachgewiesen, dass die subzelluläre Lokalisation dieser Deletionsmutante der des endogenen APP entspricht. Die Sekretion dieses stark verkürzten sAPP erfolgte wie die des Wildtyps basolateral. Dieses Ergebnis deutet an, dass die Determinante für die basolaterale Sekretion des sAPP nicht innerhalb der Ektodomäne liegt, wie in einigen älteren Publikationen angenommen wird [Haass et al. 1995, de Strooper et al. 1995]. Neuere Ergebnisse dagegen führen die polare Sekretion des sAPP auf die basolaterale Lokalisation der α-Sekretase zurück [Capell et al. 2002], was die basolaterale Sekretion der Deletionsmutante erklären könnte.
sAPP-Bindung an polaren Zellen
Durch Interaktion mit einem bisher unbekannten Rezeptorprotein erfüllt sAPP für verschiedene Zelltypen die Funktion eines Wachstumsfaktors [Saitoh et al., 1989, Pietrzik et al., 1998, Hoffmann et al., 2000]. Da viele Wachstumsfaktor-Rezeptoren selektiv entweder an der apikalen oder basolateralen Plasmamembran von Epithelzellen lokalisiert sind, wurden Bindungsstudien mit rekombinant exprimiertem sAPP (sAPPrec) an polaren FRT und MDCK-Zellen durchgeführt. Analyse der Bindung mit einem sAPPrec-spezifischen Antikörper zeigte, dass sAPP ausschließlich an der apikalen Plasmamembran beider Zelllinien bindet. Da die Sekretion des sAPP in FRT-Zellen ebenso apikal erfolgt, ist in dieser Zelllinie eine autokrine Regulation durch sAPP vorstellbar, was auch durch vorherige Ergebnisse angedeutet wurde [Pietrzik et al. 1998]. Für MDCK-Zellen, die sAPP basolateral sezernieren und apikal binden, muss ein anderer Regulationsmechanismus vorliegen. Es könnte sich um parakrine Regulation handeln, was jedoch noch bestätigt werden muss.
Fazit: In dieser Arbeit wurde zum ersten Mal gezeigt, dass tetramere Adaptor-ProteinKomplexe eine Rolle im intrazellulären Transport von APP spielen. In diesem Zusammenhang wurde die Funktion des AP-4-Komplexes in einer Kollaboration analysiert. Es wurde gezeigt, dass AP-1A und AP-1B eine Rolle im Transport von APP spielen. Eine Funktion von AP-4 im Transport von APP ist nach den vorliegenden Ergebnissen unwahrscheinlich. Untersuchungen zur APP-Sortierung in verschiedenen Epithelzelllinien zeigten, dass die Hypothese der Unabhängigkeit von APP-Transport und sAPP-Sekretion als genereller Mechanismus angesehen werden kann. Durch Analyse der sAPP-Bindung an polaren FRT- und MDCK-Zellen wurde erstmals die polare Lokalisation des putativen sAPP-Rezeptors untersucht, was einen ersten Einblick in den Mechanismus der sAPP-vermittelten Regulation in polaren Zellen ermöglichte.
Cytochrome P450 (CYP) enzymes oxidize, peroxidize and/or reduce cholesterol, vitamins, steroids, xenobiotics and numerous pharmacological substances in an oxygen- and NADPHdependent manner. Since many CYP isozymes are also capable of metabolizing arachidonic acid to biologically active products, CYP enzymes are often described as the third pathway of arachidonic acid metabolism i.e., in addition to cyclooxygenases and lipoxygenases. CYP enzymes are predominantly expressed in the liver while others, such as members of the CYP 2J, CYP 2C and CYP 4A subfamilies, can be detected in extrahepatic tissues, particularly in the cardiovascular system. Recent data suggest that a CYP 2C enzyme(s) expressed in coronary artery endothelial cells generate epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12- and 14,15-EET) which contribute to the acute control of vascular tone and the longterm regulation of vascular homeostasis.
The expression of CYP 2C in coronary artery endothelial cells is regulated by a number of stimuli, such as cyclic stretch and fluid shear stress as well as by the corticosteroid cortisol and a number of CYP substrates (nifedipine, cerivastatin and -naphthoflavone). However, the signalling pathways and the transcription factors involved in regulating the expression of the gene are unknown.
Since most of the CYP 2C enzymes are transcriptionally regulated, we were interested in identifying the CYP 2C isoform(s) expressed in porcine coronary artery endothelial cells (PCAEC) as well as determining its/their promoter sequence(s). The overall goal was to study the involvement of different transcription factor binding elements in the regulation of the CYP 2C gene(s). Porcine coronary arteries were used given the possibility of analysing the results obtained at the cellular level with alterations in vascular function. Comparison of the porcine CYP 2C and the human CYP 2C8 and 2C9 promoters was also a major goal of this study.
To identify the relevant porcine CYP 2C isoform nested RT-PCR was performed using total RNA from porcine coronary artery endothelial cells. Comparison of the sequence of the product of this reaction with the NCBI database suggested that the CYP 2C expressed in PCAEC was approximately 85% homologous with the human CYP 2C9 enzyme. To obtain the full length CYP 2C isoform 5´ rapid amplification of cDNA end (5´ RACE) was performed using a downstream reverse gene specific primer which is conserved in all of the porcine CYP 2C isoforms. The intention behind using such a primer was to amplify all the possible CYP cDNAs expressed in PCAEC. With the 5´ RACE technology it was possible not only to identify the exact isoform (CYP 2C34) expressed in PCAEC, but it was also possible to amplify 550 bp of the 5´ upstream region. This result was authenticated by comparing the protein/nucleotide sequence with other human CYP 2C genes such as CYP 2C8 and CYP 2C9 as well as different porcine CYP 2C genes (CYP 2C34, CYP 2C49). Multiple protein/nucleotide sequence alignment revealed approximately 85-90% sequence identity. An exon1-2 specific radio-labelled probe of the CYP 2C34 gene was then used to screen a porcine genomic library for positive genomic clones containing the promoter region of the CYP 2C34 gene.
For the isolation of 5´ flanking region of CYP 2C34 gene a PCR-based directional genome walking strategy was used in which the positive porcine genomic BAC clones were taken as a DNA template. Four arbitrarily designed universal walking primers and a gene-specific primer derived from the CYP 2C34 gene sequence were employed and led to the identification and isolation of 1.4 kb of the 5´ flanking region.
The 1.4 kb 5´ flanking region of CYP 2C34 gene contains multiple transcription factor binding sites including glucocorticoid-responsive element (GRE), hypoxia-responsive element (HRE), CAAT-enhancer binding protein (C/EBP), stress responsive element (STRE) consensus sequences. CYP 2C34 promoter constructs were generated and reporter gene activity (luciferase) activity was compared with that of a promoterless vector (pGL3-Basic) at first in HEK cells and then in PCAEC. After using cortisol as a positive control to demonstrate that the promoter constructs generated were functional we determined the effects of physiologically relevant stimuli i.e., hypoxia and cyclic stretch. Additional experiments with zinc sulphate were performed in a preliminary analysis of the role of Zn2+ inducible transcription factors and might be cooperative heterodimerization formation with these transcription factor with C/EBP in the regulation of CYP 2C34 expression. With all these stimuli, reporter gene activity of CYP 2C34 promoter was significantly (3-8 fold) increased over values obtained in unstimulated cells.
Analysis of the regions that are essential for the induction of promoter activity in response to the different stimuli of interest have to be performed in combination with gel shift assays, siRNA experiments as well as site-directed mutagenesis experiments. Comparison of the regulation of the CYP 2C34 gene and correlation with changes in vascular function (in isolated porcine coronary arteries) should deliver information relevant to the regulation of the CYP 2C enzyme expressed in human coronary artery endothelial cells. The recent demonstration of a clinically relevant role for CYP 2C9 in coronary heart disease underlines the importance of such a study.
Determination of the structure of complex I of Yarrowia lipolytica by single particle analysis
(2004)
Komplex I enthält ein Flavinmononukleotid sowie mindestens acht Eisen- Schwefel Zentren als redoxaktive Cofaktoren. Da ein wesentlicher Teil des mitochondrialen Genoms für Untereinheiten von Komplex I codiert, betrifft eine Vielzahl von mitochondrialen Erkrankungen diesen Enzymkomplex.
Komplex I wurde bisher aus Mitochondrien, Chloroplasten und Bakterien isoliert. Die Minimalform von Komplex I wird in Bakterien gefunden, wo er aus 14 (bzw 13 im Falle einer Genfusion) Untereinheiten besteht und eine Masse von etwa 550 kDa aufweist. Generell werden sieben hydrophile und sieben hydrophobe Untereinheiten mit über 50 vorhergesagten Transmembranhelices gefunden. Im Komplex I aus Eukaryoten wurde eine grössere Anzahl zusätzlicher, akzessorischer Untereinheiten nachgewiesen. Hier werden die sieben hydrophoben Untereinheiten vom mitochondrialen Genom codiert, während alle anderen Untereinheiten kerncodiert sind und in das Mitochondrium importiert werden müssen.
Die obligat aerobe Hefe Yarrowia lipolytica wurde als Modellsystem zur Untersuchung von eukaryotischem Komplex I etabliert. Die bisher am besten untersuchte Hefe Saccharomyces cerevisiae enthält keinen Komplex I. Hier wird die Oxidation von NADH durch eine andere Klasse von sogenannten alternativen NADH Dehydrogenasen durchgeführt. Auch Y. lipolytica enthält ein solches alternatives Enzym, das allerdings mit seiner Substratbindungsstelle zur Aussenseite der inneren Mitochondrienmembran orientiert ist. Durch molekularbiologische Manipulation konnte eine interne Version dieses Enzymes exprimiert werden, wodurch es möglich ist, letale Defekte in Komplex I Deletionsmutanten zu kompensieren. Mittlerweile wurden alle Voraussetzungen geschaffen, um kerncodierte Untereinheiten von Komplex I aus Y. lipolytica gezielt genetisch zu verändern. Die Proteinreinigung wird durch die Verwendung einer auf einem His-tag basierenden Affinitätsreinigung erheblich erleichtert...
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Highlights
• Cryo-EM structure of a yeast F1Fo-ATP synthase dimer
• Inhibitor-free X-ray structure of the F1 head and rotor complex
• Mechanism of ATP generation by rotary catalysis
• Structural basis of cristae formation in the inner mitochondrial membrane
Summary
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.
Understanding the complexity of transcriptional regulation is a major goal of computational biology. Because experimental linkage of regulatory sites to genes is challenging, computational methods considering epigenomics data have been proposed to create tissue-specific regulatory maps. However, we showed that these approaches are not well suited to account for the variations of the regulatory landscape between cell-types. To overcome these drawbacks, we developed a new method called STITCHIT, that identifies and links putative regulatory sites to genes. Within STITCHIT, we consider the chromatin accessibility signal of all samples jointly to identify regions exhibiting a signal variation related to the expression of a distinct gene. STITCHIT outperforms previous approaches in various validation experiments and was used with a genome-wide CRISPR-Cas9 screen to prioritize novel doxorubicin-resistance genes and their associated non-coding regulatory regions. We believe that our work paves the way for a more refined understanding of transcriptional regulation at the gene-level.
In fungi, the mitochondrial respiratory chain complexes (complexes I–IV) are responsible for oxidative phosphorylation, as in higher eukaryotes. Cryo-EM was used to identify a 200 kDa membrane protein from Neurospora crassa in lipid nanodiscs as cytochrome c oxidase (complex IV) and its structure was determined at 5.5 Å resolution. The map closely resembles the cryo-EM structure of complex IV from Saccharomyces cerevisiae. Its ten subunits are conserved in S. cerevisiae and Bos taurus, but other transmembrane subunits are missing. The different structure of the Cox5a subunit is typical for fungal complex IV and may affect the interaction with complex III in a respiratory supercomplex. Additional density was found between the matrix domains of the Cox4 and Cox5a subunits that appears to be specific to N. crassa.
CryoEM at IUCRJ: a new era
(2016)
Highlights
• Cryo-EM structures of the yeast low-affinity phosphate importer ScPho90
• Complementary structures reveal insights into the substrate translocation mechanism
• Comparisons with homologous transporters highlight the conserved transport mechanism
• Regulation by the SPX domain is discussed
Summary
Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.