Kaiserslautern - Fachbereich Biologie
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Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a “transport metabolon” with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3– and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme’s catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.
Biological clocks exist across all life forms and serve to coordinate organismal physiology with periodic environmental changes. The underlying mechanism of these clocks is predominantly based on cellular transcription-translation feedback loops in which clock proteins mediate the periodic expression of numerous genes. However, recent studies point to the existence of a conserved timekeeping mechanism independent of cellular transcription and translation, but based on cellular metabolism. These metabolic clocks were concluded based upon the observation of circadian and ultradian oscillations in the level of hyperoxidized peroxiredoxin proteins. Peroxiredoxins are enzymes found almost ubiquitously throughout life. Originally identified as H2O2 scavengers, recent studies show that peroxiredoxins can transfer oxidation to, and thereby regulate, a wide range of cellular proteins. Thus, it is conceivable that peroxiredoxins, using H2O2 as the primary signaling molecule, have the potential to integrate and coordinate much of cellular physiology and behavior with metabolic changes. Nonetheless, it remained unclear if peroxiredoxins are passive reporters of metabolic clock activity or active determinants of cellular timekeeping. Budding yeast possess an ultradian metabolic clock termed the Yeast Metabolic Cycle (YMC). The most obvious feature of the YMC is a high amplitude oscillation in oxygen consumption. Like circadian clocks, the YMC temporally compartmentalizes cellular processes (e.g. metabolism) and coordinates cellular programs such as gene expression and cell division. The YMC also exhibits oscillations in the level of hyperoxidized peroxiredoxin proteins.
In this study, I used the YMC clock model to investigate the role of peroxiredoxins in cellular timekeeping, as well as the coordination of cell division with the metabolic clock. I observed that cytosolic 2-Cys peroxiredoxins are essential for robust metabolic clock function. I provide direct evidence for oscillations in cytosolic H2O2 levels, as well as cyclical changes in oxidation state of a peroxiredoxin and a model peroxiredoxin target protein during the YMC. I noted two distinct metabolic states during the YMC: low oxygen consumption (LOC) and high oxygen consumption (HOC). I demonstrate that thiol-disulfide oxidation and reduction are necessary for switching between LOC and HOC. Specifically, a thiol reductant promotes switching to HOC, whilst a thiol oxidant prevents switching to HOC, forcing cells to remain in LOC. Transient peroxiredoxin inactivation triggered rapid and premature switching from LOC to HOC. Furthermore, I show that cell division is normally synchronized with the YMC and that deletion of typical 2-Cys peroxiredoxins leads to complete uncoupling of cell division from metabolic cycling. Moreover, metabolic oscillations are crucial for regulating cell cycle entry and exit. Intriguingly, switching to HOC is crucial for initiating cell cycle entry whilst switching to LOC is crucial for cell cycle completion and exit. Consequently, forcing cells to remain in HOC by application of a thiol reductant leads to multiple rounds of cell cycle entry despite failure to complete the preceding cell cycle. On the other hand, forcing cells to remain in LOC by treating with a thiol oxidant prevents initiation of cell cycle entry.
In conclusion, I propose that peroxiredoxins – by controlling metabolic cycles, which are in turn crucial for regulating the progression through cell cycle – play a central role in the coordination of cellular metabolism with cell division. This proposition, thus, positions peroxiredoxins as active players in the cellular timekeeping mechanism.
Untersuchungen zur Struktur und Spezifität der Phycobiliproteinlyase CPES aus Guillardia theta
(2020)
Cryptophyten verwenden neben Chlorophyll zusätzliche Lichtsammelproteine für die Photo-synthese – die Phycobiliproteine (PBP). In Cyanobakterien, Rotalgen und Glaukophyten sind PBP ebenfalls ubiquitär verbreitet. Für den Zweck der Lichtsammlung tragen die PBP- Untereinheiten kovalent gebundene offenkettige Tetrapyrrol-Chromophore an konservierten Cysteinresten. Diese Phycobiline sind in der Lage, grünes Licht zu absorbieren und es für die Photosynthese zur Verfügung zu stellen. Die Fähigkeit zur Photosynthese erlangten Crypto-phyten bei der sekundären Endosymbiose durch Aufnahme einer früheren Rotalge. Die evolutionäre Entwicklung brachte schließlich modifizierte PBP hervor. In Gegensatz zu ande-ren Organismen liegen die PBP in Cryptophyten in löslicher Form im Thylakoidlumen des Plastiden vor. Cryptophyten besitzen lediglich einen Typ an PBP, Guillardia theta verwendet Phycoerythrin PE545. Die α-Untereinheiten sind jeweils mit einem Molekül 15,16-Dihydrobi-liverdin (DHBV) und die β-Untereinheiten mit drei Molekülen Phycoerythrobilin (PEB) chromophoryliert. Die Chromophorylierung cryptophytischer Apo-PBP ist bisher wenig un-tersucht und verstanden. Aus Cyanobakterien ist jedoch bekannt, dass die Chromophorylie-rung häufig mit Hilfe von Phycobiliproteinlyasen (PBP Lyasen) stattfindet, welche die Phyco-bilinübertragung unterstützen.
In der vorliegenden Arbeit erfolgte die funktionelle Charakterisierung der eukaryotischen S-Typ-PBP Lyase GtCPES aus G. theta. Mittels Fluoreszenzspektroskopie und Zink-induzierter Fluoreszenz konnte gezeigt werden, dass GtCPES den Transfer von 3(Z)-PEB auf Cys82 der PBP-β-Untereinheit aus Prochlorococcus marinus MED4 (PmCpeB) vermittelt. An der PEB-Bindung sowie am -Transfer beteiligte Aminosäuren wurden mit Hilfe Zielgerichteter Muta-genese identifiziert. Anhand spektroskopischer Binde- und Transferstudien mit den Protein-varianten wurden drei Aminosäuren in der Ligandenbindetasche ermittelt, die relevant für die Bindung sind (Trp69, Glu136, Glu168). Diese koordinieren vermutlich PEB in der Bindetasche und stabilisieren somit die Konformation. Zusätzlich konnten zwei im PEB-Transfer involvierte Aminosäuren eindeutig identifiziert werden (Trp75, Ser150). Trp75 kommt dabei eine essenzielle Bedeutung für den Transfer zu. Des Weiteren konnte gezeigt werden, dass Met67 für die auf PEB und DHBV beschränkte Substratspezifität von GtCPES verantwortlich ist. Die Variante GtCPES_M67A bindet sowohl PEB als auch das rigide Phycocyanobilin (PCB) stabil unter Bildung eines farbigen Komplexes in vitro und in vivo in Escherichia coli. GtCPES_M67A scheint zudem in der Lage zu sein, PCB auf geeignete Apo-Proteine zu transfe-rieren. Neben der sterischen und elektrostatischen Umgebung entscheidet damit zusätzlich die Substratspezifität der PBP Lyase über die gebundenen Chromophore am PBP.
In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.
Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive—a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions
The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.
We isolated an encysted ciliate from a geothermal field in Iceland. The morphological features of this isolate fit the descriptions of Dexiotricha colpidiopsis (Kahl, 1926) Jankowski, 1964 very well. These comprise body shape and size in vivo, the number of somatic kineties, and the positions of macronucleus and contractile vacuole. Using state-of-the-art taxonomic methods, the species is redescribed, including phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) gene as molecular marker. In the phylogenetic analyses, D. colpidiopsis clusters with the three available SSU rRNA gene sequences of congeners, suggesting a monophyly of the genus Dexiotricha. Its closest relative in phylogenetic analyses is D. elliptica, which also shows a high morphological similarity. This is the first record of a Dexiotricha species from a hot spring, indicating a wide temperature tolerance of this species at least in the encysted state. The new findings on D. colpidiopsis are included in a briefly revision of the scuticociliate genus Dexiotricha and an identification key to the species.
Słowa kluczowe: Dexiotricha, hot spring, morphology, phylogeny, SSU rRNA gene
Biological soil crusts (biocrusts) are a common element of the Queensland (Australia) dry savannah ecosystem and are composed of cyanobacteria, algae, lichens, bryophytes, fungi and heterotrophic bacteria. Here we report how the CO2 gas exchange of the cyanobacteria-dominated biocrust type from Boodjamulla National Park in the north Queensland Gulf Savannah responds to the pronounced climatic seasonality and on their quality as a carbon sink using a semi-automatic cuvette system. The dominant cyanobacteria are the filamentous species Symplocastrum purpurascens together with Scytonema sp. Metabolic activity was recorded between 1 July 2010 and 30 June 2011, during which CO2 exchange was only evident from November 2010 until mid-April 2011, representative of 23.6 % of the 1-year recording period. In November at the onset of the wet season, the first month (November) and the last month (April) of activity had pronounced respiratory loss of CO2. The metabolic active period accounted for 25 % of the wet season and of that period 48.6 % was net photosynthesis (NP) and 51.4 % dark respiration (DR). During the time of NP, net photosynthetic uptake of CO2 during daylight hours was reduced by 32.6 % due to water supersaturation. In total, the biocrust fixed 229.09 mmol CO2 m−2 yr−1, corresponding to an annual carbon gain of 2.75 g m−2 yr−1. Due to malfunction of the automatic cuvette system, data from September and October 2010 together with some days in November and December 2010 could not be analysed for NP and DR. Based on climatic and gas exchange data from November 2010, an estimated loss of 88 mmol CO2 m−2 was found for the 2 months, resulting in corrected annual rates of 143.1 mmol CO2 m−2 yr−1, equivalent to a carbon gain of 1.7 g m−2 yr−1. The bulk of the net photosynthetic activity occurred above a relative humidity of 42 %, indicating a suitable climatic combination of temperature, water availability and light intensity well above 200 µmol photons m−2 s−1 photosynthetic active radiation. The Boodjamulla biocrust exhibited high seasonal variability in CO2 gas exchange pattern, clearly divided into metabolically inactive winter months and active summer months. The metabolic active period commences with a period (of up to 3 months) of carbon loss, likely due to reestablishment of the crust structure and restoration of NP prior to about a 4-month period of net carbon gain. In the Gulf Savannah biocrust system, seasonality over the year investigated showed that only a minority of the year is actually suitable for biocrust growth and thus has a small window for potential contribution to soil organic matter.
Die Funktion der c-di-GMP modulierenden Membranproteine NbdA und MucR in Pseudomonas aeruginosa
(2019)
NbdA und MucR sind Multi-Domänenproteine aus Pseudomonas aeruginosa. Beide Proteine besitzen eine ähnliche Domänenorganisation mit einer N-terminalen, membranständigen
MHYT-Domäne sowie einer GGDEF- und einer EAL-Domäne im Cytoplasma. Die
cytosolischen Domänen von MucR sind beide aktiv, während NbdA neben der intakten
EAL-Domäne eine degenerierte GGDEF-Domäne mit dem Motiv AGDEF aufweist. Bioinformatischen
Vorhersagen zufolge soll die MHYT-Domäne eine sensorische Funktion für diatomische Gase wie Stickstoffmonoxid oder Sauerstoff vermitteln. Die phänotypische Charakterisierung der markerlosen PAO1-Deletionsmutanten \(Delta\)nbdA, \(Delta\)mucR und \(Delta\)nbdA \(Delta\)mucR zeigte, dass NbdA und MucR nicht in die NO-induzierte
Dispersion involviert sind. Ebenso konnte in einem neu etablierten heterologen in-vivo-System in E. coli keine NO-sensorische Funktion der Proteine detektiert werden. Im Weiteren wurde festgestellt, dass die MHYT-Domäne keinen ersichtlichen Einfluss auf die
Enzymaktivität von NbdA und MucR unter aeroben Bedingungen hat. Demzufolge fungiert
die Membrandomäne vermutlich weder als Sensor für Sauerstoff, noch für NO. Anhand heterologer Komplementationstests konnte eine PDE-Aktivität des NbdA-Volllängenproteins
nachgewiesen werden. Zudem wurde gezeigt, dass die degenerierte AGDEF-Domäne einen regulatorischen Effekt auf die EAL-Domäne hat, der essentiell für die in-
vivo-Aktivität von NbdA ist. In-vivo-Untersuchungen bestätigten die postulierte DGC-Aktivität von MucR. Weiterhin konnte belegt werden, dass MucR ein bifunktionelles Enzym
ist. Entgegen den Erwartungen scheint es jedoch im Planktonischen als DGC und im
Biofilm als PDE zu fungieren.
Ein weiterer Aspekt dieser Arbeit war die Charakterisierung der homologen Überexpression
von nbdA in P. aeruginosa, welche teilweise unerwartete Phänotypen ergab. Anhand
der homologen Überproduktion einer inaktiven NbdA-Variante stellte sich heraus, dass die
Hemmung der Motilität unabhängig von der Aktivität von NbdA auftritt. Massenspektrometrische
Analysen deuteten daraufhin, dass NbdA lokal c-di-GMP hydrolysiert. Diese
Ergebnisse implizieren, dass NbdA eine Trigger-PDE ist, deren primäre Funktion die Regulation
anderer makromolekularer Zielmoleküle ist. In Pseudomonas fluorescens Pf0-1 ist bekannt, dass das NbdA-Homolog Pfl01_1252 mit den Homologen von MucR (Pfl01_2525) und SadC (Pfl01_4451) interagiert. Ergebnisse einer früheren Arbeit lassen eine Interaktion
von NbdA und SadC ebenso in P. aeruginosa vermuten. Daher ist denkbar, dass sich
NbdA im gleichen Netzwerk wie MucR und SadC befindet und deren Aktivität reguliert.
Linking protistan community shifts along salinity gradients with cellular haloadaptation strategies
(2019)
Salinity is one of the most structuring environmental factors for microeukaryotic communities. Using eDNA barcoding, I detected significant shifts in microeukaryotic community compositions occurring at distinct salinities between brackish and marine conditions in the Baltic Sea. I, furthermore, conducted a metadata analysis including my and other marine and hypersaline community sequence data to confirm the existence of salinity-related transition boundaries and significant changes in alpha diversity patterns along a brackish to hypersaline gradient. One hypothesis for the formation of salinity-dependent transition boundaries between brackish to hypersaline conditions is the use of different cellular haloadaptation strategies. To test this hypothesis, I conducted metatranscriptome analyses of microeukaryotic communities along a pronounced salinity gradient (40 – 380 ‰). Clustering of functional transcripts revealed differences in metabolic properties and metabolic capacities between microeukaryotic communities at specific salinities, corresponding to the transition boundaries already observed in the taxonomic eDNA barcoding approach. In specific, microeukaryotic communities thriving at mid-hypersaline conditions (≤ 150 ‰) seem to predominantly apply the ‘low-salt – organic-solutes-in’ strategy by accumulating compatible solutes to counteract osmotic stress. Indications were found for both the intracellular synthesis of compatible solutes as well as for cellular transport systems. In contrast, communities of extreme-hypersaline habitats (≥ 200 ‰) may preferentially use the ‘high-salt-in’ strategy, i. e. the intracellular accumulation of inorganic ions in high concentrations, which is implied by the increased expression of Mg2+, K+, Cl- transporters and channels.
In order to characterize the ‘low-salt – organic-solutes-in’ strategy applied by protists in more detail, I conducted a time-resolved transcriptome analysis of the heterotrophic ciliate Schmidingerothrix salinarum serving as model organism. S. salinarum was thus subjected to a salt-up shock to investigate the intracellular response to osmotic stress by shifts of gene expression. After increasing the external salinity, an increased expression of two-component signal transduction systems and MAPK cascades was observed. In an early reaction, the expression of transport mechanisms for K+, Cl- and Ca2+ increased, which may enhance the capacity of K+, Cl- and Ca2+ in the cytoplasm to compensate possibly harmful Na+ influx. Expression of enzymes for the synthesis of possible compatible solutes, starting with glycine betaine, followed by ectoine and later proline, could imply that the inorganic ions K+, Cl- and Ca2+ are gradually replaced by the synthesized compatible solutes. Additionally, expressed transporters for choline (precursor of glycine betaine) and proline could indicate an intracellular accumulation of compatible solutes to balance the external salinity. During this accumulation, the up-regulated ion export mechanisms may increase the capacity for Na+ expulsion from the cytoplasm and ion compartmentalization between cell organelles seem to happen.
The results of my PhD project revealed first evidence at molecular level for the salinity-dependent use of different haloadaptation strategies in microeukaryotes and significantly extend existing knowledge about haloadaptation processes in ciliates. The results provide ground for future research, such as (comparative) transcriptome analysis of ciliates thriving in extreme-hypersaline habitats or experiments like qRT-PCR to validate transcriptome results.