Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

This dissertation addresses the biogeographic history of the Araceae family and of one of its largest genera, Alocasia. With >3300 species, Araceae are among the largest families of flowering plants. It is the monocot lineage with the deepest fossil record, reaching back to the Early Cretaceous. Araceae are distributed worldwide, but >3100 species occur in the tropical regions of the Americas, Asia, Africa, and Australia; most fossils from the Late Cretaceous and many younger ones come from the temperate zone in the northern hemisphere, implying much extinction and range expansion. Most subfamilies are pantropically distributed, and almost all genera are restricted to one continent. Alocasia comprises 113 species, many as yet undescribed, making it the 7 th -largest genus of the Araceae. Many species are ornamentals, and two species are of interest for man, either for food (giant taro) or in local cultures (Chinese taro). The origin of these species was not known. Alocasia is distributed in Southeast Asia from India to Australia, with species occurring on all islands of the Malay Archipelago. This region has a complex geologic history shaped by the collision of the Eurasian, the Pacific, and the Indo-Australian plate. The Malesian flora and fauna comprises Laurasian and Gondwanan elements, reflecting the influence of changing sea levels, uplift and submergence of islands, and other tectonic movement. In this thesis, I used molecular phylogenetics, Bayesian divergence dating, ancestral area reconstruction to understand the past distribution of the Araceae family and the Alocasia clade in the context of past continent movements and climate history. For the family analysis, existing chloroplast DNA matrices were augmented so that all Araceae genera were represented by one or more species, with a focus on covering geographic disjunctions, especially between continents. Divergence dating relied on seven confidently assigned fossil constraints, comparing uniform and gamma-shaped prior distributions on fossil ages, as well as several molecular clock models. Biogeographic analyses were performed in a model-based likelihood framework that took into account past dispersal routes based on continent connectivity and climate. I also integrated fossils into the ancestral area reconstruction, either simulating extinct or still existing ranges, and then compared results to those obtained from analyses without fossils. To study the morphology and ecology of Alocasia, fieldwork was conducted in Malaysia and herbarium work in Germany, the Netherlands, Indonesia, Malaysia, and Singapore. Maximum likelihood phylogenies were inferred based on chloroplast and nuclear loci, sequenced for 71 species of Alocasia plus 25 outgroup species from 16 genera. Bayesian divergence dating of the nuclear phylogeny relied on one fossil constraint and ancestral areas were reconstructed using parsimony- and likelihood-based methods. The Araceae diverged from the remaining Alismatales in the Early Cretaceous (ca. 135 Ma ago), and all eight subfamilies originated before the Cenozoic. The earliest lineages are inferred to have occurred in Laurasia (based on fossils and tree topology), and most lineages reached Africa, South America, Southeast Asia, and Australia during the Paleogene and Neogene. Many clades experienced extinction in the temperate regions of the northern hemisphere during the Oligocene climate cooling. Two continentally disjunct genera (Nephthytis and Philodendron) are polyphyletic and need taxonomic rearrangement. Plastid substitution rates are exceptionally high in free-floating and water-associated Araceae. Ancestral area reconstructions obtained when fossil (no longer occupied) ranges where included in the analyses were more plausible than those without fossil ranges. This is not a trivial result because only in a quantitative (computer-based) analysis is it possible for fossil ranges to influence results (here areas) at distant nodes in the phylogenetic tree. The nuclear and plastid phylogenies of Alocasia revealed the polyphyly of the two genera Alocasia and Colocasia; to achieve monophyly, two species (Alocasia hypnosa and Colocasia gigantea) have to be moved to other genera. There were strong incongruencies between phylogenies from the two partitions: The chloroplast data reflect geographical proximity, the nuclear morphological similarity. This may indicate hybridization events followed by chloroplast capture. Based on the nuclear tree, Alocasia split from its sister group by the end of the Oligocene (ca. 24 Ma) and colonized the Malay Archipelago from the Asian mainland. Borneo played a central role, with 11–13 of 18–19 inferred dispersal events originating there. The Philippines were reached from Borneo 4–5 times in the late Miocene and early Pliocene, and the Asian mainland 6–7 times during the Pliocene. The geographic origin of two domesticated species could be resolved: Giant taro originated on the Philippines and Chinese taro on the Asian mainland.

Das Kalzium Signalnetzwerk ist ein wichtiger Übermittler von internen und externen Reizen in der Pflanze. Diese Signale werden unter anderem durch Calmodulin und calmodulin ähnlichen Proteine an verschiedene Effektorproteine weitergeleitet. AFG1L2 (AFG1 like Protein 2) wurde in dieser Arbeit als calmodulin bindendes Proteine aus der Familie der AAA+ Proteine (ATPasen associated with various cellular activities) identifiziert. Mit Hilfe von GFP Fusionskonstrukten konnte die in vivo Lokalisierung von AFG1L2 in Mitochondrien gezeigt werden, und die duale Lokalisierung des bereits zuvor beschriebenen Homologs AFG1L1 in Chloroplasten und Mitochondrien bestätigt werden. Die Bindung von Calmodulin erfolgt bei AFG1L2 kalziumabhängig und die Calmodulin Bindedomäne befindet sich homolog zu AFG1L1 nahe dem für die ATP Hydrolyse essenziellen Walker A Motiv in der AAA Domäne. Es konnte gezeigt werden, dass ADP die Interaktion von AFG1L2 und Calmodulin verstärkt. Das weist auf einen inhibierenden Effekt von Calmodulin hin, da die Nucleotid Bindetasche besetzt bleibt und keine erneute ATP Hydrolyse stattfinden kann. Neben den konservierten Motiven haben AAA+ Proteine die Bildung von Oligomeren, meist Hexameren gemeinsam. In der Regel ist der Komplex die hydrolytisch aktive Form. Es konnte gezeigt werden, dass AFG1L2 prinzipiell in der Lage ist Oligomere zu bilden, aber dass das Vorliegen als Komplex wahrscheinlich nicht für die Hydrolyse von ATP erforderlich ist. Um die Funktion von AFG1L1 und AFG1L2 in der Pflanze zu bestimmen, wurden afg1l1 und afg1l2 Mutanten untersucht. Diese zeigten aber unter den normalen Bedingungen keinen Phänotyp. Auch eine im Zuge dieser Arbeit generierte afg1l1/afg1l2 Doppel Mutante wies unter Standart-Bedingungen keine Beeinträchtigungen im Wachstum auf. Analysen der Expression der Proteine legen nahe, dass AFG1L1 und AFG1L2 unter Gewächshaus Bedingungen einander nicht substituieren. DNA Microarray Daten lassen jedoch auf eine Beteiligung von AFG1L1 an der Stress Toleranz gegen Salz und Hyper-Osmolarität schließen.

Oxygenic photosynthesis converts light energy into chemical energy and is responsible for generating most of our atmosphere’s oxygen and biomass on earth. Several multimeric protein complexes are involved in the underlying photosynthetic electron transfer chain with photosystem II (PSII) representing the initial complex mediating the extraction of electrons from water molecules, thus generating molecular oxygen as a by-product. During recent years, the structural details and components of the PSII complex, including its inorganic and organic cofactors, have been elucidated in great detail. However, little is known about the assembly pathway of this at least 20 protein subunits containing machinery. Previous work indicated that PSII assembly occurs in a step-wise fashion and requires a number of facilitating factors, which interact transiently with nascent PSII complexes. Earlier studies of one of those assembly factors, the cyanobacterial PratA protein, suggested that PSII biogenesis does not only underlie a temporal order but is also organized at the spatial level, as PratA was shown to mark a special intermediate membrane subfraction (PDMs), hypothesized to represent regions for initial steps of PSII biogenesis. The presented work focused on a more detailed characterization of PDMs, clearly supporting their significance not only with regard to early protein assembly, but also concerning pigment synthesis and integration into the PSII precomplexes. The PDMs could further be allocated to special membrane regions, named biogenesis centers, at sites where thylakoid membranes converge to the plasma membrane, thus demonstrating the spatial organization of PSII assembly at the cellular level. Moreover, a novel function of PratA in preloading of PSII with Mn2+ ions, necessary for construction of the water-splitting complex, was discovered. Concomitantly with progression of the assembly, the nascent PSII complexes are transported from the PDMs to the thylakoid membrane system, where Sll0933 – a novel PSII assembly factor identified in this thesis – mediates the integration of the PSII inner antenna proteins followed by completion of the assembly process. Additionally, it could be shown that many of the so far identified facilitating factors interact with each other and, thus, form a complex network for PSII assembly. Especially the interaction between the two assembly factors YCF48 and Sll0933 was characterized in more detail, revealing a successive mode of action with YCF48 operating upstream of Sll0933. Taken together, the presented results enable the development of an extended and elaborated model of PSII assembly, which is a concerted process connecting protein and cofactor synthesis/integration in a spatiotemporal manner, and thus contribute to a more profound understanding of photosynthesis itself.

Ein großer Teil der Fragestellungen in den Neurowissenschaften beschäftigt sich mit dem Thema, wie das Säugerhirn Verhalten auslöst und steuert. Die Schreckreaktion ist ein relativ einfaches Verhalten, welches bei Säugetieren ohne großen Aufwand ausgelöst werden kann und variabel auf eine Vielfalt von experimentellen Behandlungen reagiert. Das Ziel der vorliegenden Arbeit war es, Schreckreaktions-Messungen am Max-Planck- Institut für Psychiatrie in München (MPI-P) zu etablieren. Vor dem Hintergrund aktueller Fragestellungen sollten die Experimente zu einsatzbereiten Messmethoden und Verhaltensparadigmen führen. In der vorliegenden Arbeit gelang es nicht, das Paradigma der furchtpotenzierten Schreckreaktion (FPS) zuverlässig in einem häufig am MPI-P eingesetzten Mausstamm anzuwenden. Das FPS maskierende Phänomen, daß die Präsentation eines unkonditionierten Tons bereits zu einer deutlich verstärkten Schreckreaktion in diesen Mäusen führt ("tone enhanced startle", TES) wurde dann charakterisiert und im Folgenden als ergänzendes Paradigma zur Messung und Abschätzung des Hörvermögens, der Stimulus Adaptation und der Aufmerksamkeit in Mäusen vorgeschlagen. Eine Literaturrecherche ergab, daß im Paradigma der Furchtkonditionierung ("fear conditioning", FC) und deren aktives Verlernen ("extinction of FC", ExFC) verwendete Stimulus-Parameter eine hohe Varianz zwischen verschiedenen Laboratorien aufweisen. Der im Verhalten ausgelesene Lernerfolg während einer FC wie auch einem ExFC hingen in den vorliegenden Experimenten wesentlich von der verwendeten Stimulusqualität ab (d.h. sinus-Ton oder weißes Rauschen). Im Umkehrschluß empfiehlt die vorliegende Arbeit einen überlegteren Umgang mit den eingetzten Stimulus-Parametern. Es zeigte sich, daß eine erhöhte Schreckreaktion (Übererregbarkeit) ohne weiteres in einem Tiermodell der Posttraumatischen Belastungsstörung ("posttraumatic stress disorder",PTSD) gemessen werden kann. Im Weiteren konnte gezeigt werden, daß verändertes Hippocampus-Volumen in diesen Tieren, gemessen über ultramikroskopische Aufnahmen und analog zu Hippocampusveränderungen in Patienten, unabhängig von anderen PTSD-ähnlichen Symptomen dieser Mäuse ist. In einem weiteren Abschnitt widmet sich die vorliegende Arbeit der laufenden Charakterisierung der Rolle von Dopaminrezeptoren (DR) in der Präpulsinhibition (PPI) und -Faszilitierung (PPF) der Schreckreaktion. Durch lokale injektion von DR-Antagonisten konnte gezeigt werden, daß die Blockade von DR1 wiederholbar PPI verstärkt, während die Rolle von DR2, getestet mit zwei verschiedenen Antagonisten, als ambivalent gedeutet werden muß. Basierend auf diesen Experimenten wurden optogenetische Methoden in die Schreckreaktionsmessung eingeführt. Transgenen Mäusen, die lichtsensitive Ionenkanäle in ihren neuronalen Zellmembranen bestimmter Zellpopulationen tragen, wurden Lichtblitze ins Gehirn appliziert. Auf diese Weise konnten PPI und PPF unabhängig voneinander manipuliert werden. Daraus folgend, und im Unterschied zur populären Summationshypothese der PPF, schlägt die vorliegende Arbeit einen eigenständigen, von der PPI unabhängigen PPF-Schaltkreis vor, der Pyramidenneuronen der präfrontalen Kortexschicht V beinhaltet. Die vorliegende Arbeit konnte erfolgreich verschiedene Protokolle und Verhaltensparadigmen der Schreckreaktionsmessung am MPI-P etablieren und zur sofortigen Nutzung zur Verfügung stellen. Es wurden nicht nur neue Techniken wie z.B. optogenetische Methoden in die Schreckreaktionsmessung eingeführt, die vorliegenden Experiemente leisten auch ihren Beitrag zur aktiven Forschung, in dem sie z.B. die große Bedeutung der Stimulus-Parameter für den Lernerfolg von Versuchstieren nachweisen.

The following work is shedding light on the phylogenetic classification on the family of the Chlorobiacea, the members of which are showing signs of preadaptation to symbiosis. Symbioses consisting of purely prokaryotic associations between phylogenetically distinct bacterial species have been widely documented. Only few are available as a laboratory culture to elucidate the molecular basis of their interaction. One of these few model organisms is the phototrophic consortium “Chlorochromatium aggregatum”. It consists of 12-20 green sulfur bacteria epibionts surrounding a central, Betaproteobacterium in a highly ordered fashion. The phototrophic partner bacterium, belonging to the green sulfur bacteria, is available in pure culture and its physiology has been studied in detail. In this work, novel insights into the physiology of the central bacterium that was previously uncharacterized are provided. The family of the Chlorobiaceae represents a phylogenetically coherent and isolated group within the domain Bacteria. Green sulfur bacteria are obligate photolithoautotrophs that require highly reducing conditions for growth and can utilize only a very limited number of carbon substrates. These bacteria thus inhabit a very narrow ecologic niche. For the phylogenetic studies on green sulfur bacteria, 323 16S rRNA gene sequences, including cultured species as well as environmental sequences were analysed. By rarefaction analysis and statistical projection, it was shown that the data represent nearly the whole spectrum of green sulfur bacterial species that can be found in the sampled habitats. Sequences of cultured species, however, did not even cover half of the biodiversity. In the 16S rDNA gene tree, different clusters were found that in most cases correlated with physiological adaptations of the included species. By combining all sampling sites of green sulfur bacteria in a world map, large, unsampled areas were revealed and it could be shown that in some regions, a non-random distribution of GSB occurred. The wide dispersal of green sulfur bacterial species can be seen in sequences that were found ubiquitously all over the world. To imitate the phylogenetical relationships of whole genome analyses, a concatenated tree was constructed including 32 species and 3 different genetic regions, the bchG gene, the sigA gene and the fmoA gene. Comparison with the 16S rRNA gene tree showed more genetic differences between species, and led to a higher resolution and a more dependable phylogeny. A distance matrix comparison showed that the fmoA gene sequence has the highest correlation to the 16S rDNA of the sequences investigated. Additionally, a dissimilarity matrix revealed that the fmoA gene sequence provides the highest phylogenetic resolution among the sequences investigated. Therefore, we showed that the fmoA gene sequence is the most suitable among the sequences investigated to support the 16S rDNA phylogeny of green sulfur bacteria. To overcome the limitation of immobility, some green sulfur bacteria have entered into a symbiosis with motile Betaproteobacteria in a type of multicelllular association termed phototrophic consortia. Recent genomic, transcriptomic, and proteomic studies of "C. aggregatum" and its epibiont provided insights into the molecular basis and the origin of the stable association between the two very distantly related bacteria. However, to date the possibility of a metabolic coupling between the bacterial partners has not been investigated. The symbiotic exchange of metabolites between the two species was therefore investigated by tracking the flux of isotope-labeled CO2 through the two partner organisms using NanoSIMS analysis and magnetic capture, revealing a fast and simultaneous incorporation of labeled carbon into both organisms. The transferred metabolites were identified by isotopologue profiling for which the partner cells were separated by cesium chloride density gradient centrifugation, a method which identified amino acids as one group of substrates to be transferred between the two partners. The addition of external carbon substrates inhibited the transfer between the two partners, suggesting that transporters are the means by which substrates are exchanged. Genome sequencing revealed the central bacterium to be an aerobic or microaerophilic chemoheterotrophic bacterium. The existence of 32 PAS domains which are responsible for sensing various signals indicate that the central bacterium is responsible for the chemo- and phototactic responses of the consortium. The central bacterium possesses all traits of an autonomous organism. However, transcriptome analysis revealed the central bacterium to be inactive in the dark although external carbon sources were present. Thereby, a yet unexplained dependence on the epibiont is revealed which indicates a complex metabolic coupling between the two symbiotic partner organisms.

Bacteria produce and excrete signaling molecules, so called autoinducers, which allow them to monitor their population density and/or their environment in a process best known as quorum sensing. The Gram-negative marine bacterium Vibrio harveyi regulates certain virulence factors like type III secretion, siderophore production, and exoproteolytic activity as well as biofilm formation and bioluminescence using quorum sensing. The bacterium produces three different autoinducers: HAI-1, a N-(3-hydroxybutyryl)-D-homoserine lactone, AI-2, a furanosylborate diester, and CAI-1, a (Z)-3-aminoundec-2-en-4-one. The autoinducers are recognized by the hybrid sensor kinases LuxN, LuxQ and CqsS. All information is transferred to the phosphotransfer protein LuxU and the response regulator LuxO via phosphorelay and further transduced into the copy number of the master regulator LuxR. LuxR induces/represses a multitude of genes/operons (>100) including the lux-operon responsible for the production of bioluminescence. In order to understand how single cells behave within an autoinducer-activated community, autoinducer-induced processes were investigated in a homogeneous environment over time. Analysis of wild type single cells with respect to bioluminescence revealed that even at high cell densities only 70% of the cells of a population were bright. Moreover, fractionation of the population was found for autoinducer-controlled promoters (of genes coding for bioluminescence, exoproteolytic activity, and type III secretion) using reporter strains containing promoter::gfp fusions. These results indicated phenotypic heterogeneity of a genetic homogeneous population and were independent of the used cultivation medium, temperature or strain. An artificial increase of the autoinducer concentrations resulted in an all-bright cell population similar as observed for a luxO deletion mutant. Both, wild type and deletion mutant switched to biofilm formation at high cell density. However, the capability of the mutant to produce biofilm was significantly reduced. These data suggest that a population of the non-differentiating bacterium Vibrio harveyi takes advantages of division of labor. In addition, a temporal variation of the autoinducer concentrations over time was found. The extracellular concentrations of the three autoinducers and quorum sensing-regulated functions of Vibrio harveyi were monitored in a growing culture. In the early and mid-exponential growth phase only AI-2 was detectable and bioluminescence was induced. In the late exponential growth phase both, HAI-1 and AI-2 reached their maximum values, bioluminescence stayed high and exoproteolytic activity was induced. The stationary phase was characterized by equal concentrations of HAI-1 and AI-2, exoproteolytic activity reached its maximum, and CAI-1 activity was detectable in the culture fluids. Furthermore, only a stable and mature biofilm was formed, when HAI-1 and AI-2 were present in the above described ratios over time. CAI-1 had no influence on the biofilm formation in Vibrio harveyi. These results demonstrate that not the cell density per se is important, but that autoinducers rather control the development of a Vibrio harveyi population.

The formation of neuronal networks depends on the proper development and targeting of the neurons within the network. One key challenge during the development of such networks is the correct cross linking of axons and dendrites. Only correct synapse formation between dendrites and axons will allow neurons to contribute to the entire network. Therefore further insights into axon targeting mechanisms will help to understand the underlying developmental processes and contribute to future cures for a number of related diseases. Generally, once a neuron forms an axon, it starts growing towards a certain “target zone”. The underlying axon targeting mechanisms are controlled by a large number of extracellular cues provided by the extracellular matrix and neighboring cells. Depending on the neuron type, axons travel different distances towards their future synaptic partner. During that journey the neurons, more specifically the growth cone, constantly comes into contact with guidance cues. The growth cone symbolizes the forefront of an axon and is responsible to integrate different guidance signals. Depending on their nature, they trigger the local assembly or disassembly of the cytoskeleton and ultimately force the axon to turn into a certain direction. Although different guidance cues activate different signaling pathways, all of these cascades will eventually converge down on the cytoskeleton. These cytoskeletal rearrangements and changes in actin dynamics within the growth cone will promote the turning of the entire axon. In a series of events different guidance cues, attractive and repulsive, will guide the growth cone to its respective target. In this study I used the olfactory system, more specifically the olfactory receptor neurons (ORNs), of Drosophila melanogaster to investigate the mechanisms of axon targeting. The olfactory system of the fruit fly proved to be a very powerful model organism for a number of reasons: First, the number of genetic tools available for Drosophila allows the manipulation of many cellular aspects. Second, ORNs have an extremely stereotyped targeting pattern that proved to be a good system to investigate axon targeting mechanisms. The work presented in this thesis studied the role of the highly conserved actin binding protein Psidin during the development and targeting of ORNs. Herein, I was able to demonstrate that Psidin uses two independent molecular mechanisms to control ORN targeting and survival. To elucidate Psidin’s role in the aforementioned processes, I analyzed two predicted null alleles psidin1 (Brennan et al., 2007) and psidin55D4 (Kim et al., 2011), and one hypomorphic allele psidinIG978 (this study). The new hypomorphic allele psidinIG978 was mapped during this study and found to have a single point mutation within Psidin’s coding region (E320K). The data shown in this study demonstrate that Psidin is required at two different time points during the development of the olfactory system. During ORN development, Psidin is required as non-catalytic part of the N-acetyltransferase complex (NatB) to ensure ORN survival. At later stages during development, Psidin functions as an actin binding protein to regulate actin dynamics to ultimately ensure proper ORN axon targeting. I was able to show for the first time that Psidin’s previously reported function as actin binding protein in oocytes (Kim et al., 2011), is also true for neurons. The loss of Psidin leads to significantly reduced lamellipodia in growth cones of primary neurons in vitro. In agreement with Psidin’s role in actin dynamics is the finding that the parallel removal of the actin stabilizer Tropomyosin rescues the lamellipodia defect in psidin1 primary neurons. This strongly argues for Psidin being an actin destabilizing protein and antagonist of Tropomyosin. In general, psidin1 and psidin55D4 mutant axons showed severe mistargeting defects in vivo – e.g. defasciculation in Or59c and Or42a neurons or ectopic synapse formation in Or47a neurons. However, axons mutant for psidinIG978 displayed a less severe phenotype compared to the null alleles. In agreement with in vitro data, the parallel removal of Tropomyosin rescued the targeting defect in Or59c neurons in vivo. The growth cone and the lamellipodia are both important structures that keep axons responsive towards guidance cues. Therefore the lamellipodia reduction in psidin mutants is likely the cause for the observed targeting defects. Nevertheless, Psidin is required differentially among the ORN classes – the ones that project to dorsolateral or ventromedial glomeruli within the antennal lobe (AL) are more affected than centrally projecting classes. ORN classes that are more affected in psidin mutants have to turn upon entry of the AL. Therefore those classes (dorsolateral and ventromedial) have a higher requirement of Psidin, which has to maintain the lamellipodium, so that the axon can respond to cues in the first place. In addition, I overexpressed different isoforms of LimK and Cofilin to artificially create conditions that favor actin stabilization or destabilization. More generally, conditions that promoted actin destabilization and actin stabilization were able to rescue and aggravate the psidin1 phenotype, respectively. In addition to the targeting defect, psidin1 and psidin55D4 mutants showed a strong reduction in ORN cell numbers. In contrast, cell numbers were not affected in psidinIG978 mutant flies. Again, ORN classes were affected differently – e.g. Or42a neuron number was reduced by 83%, but Or59c number was only reduced by 46%. Indicating Psidin’s function in ORN survival, the expression of the anti-apoptotic protein p35 in psidin mutant neurons selectively rescued the cell number, but failed to rescue the targeting defects. Interestingly, the Psidin/Tropomyosin double mutant showed the opposite effect; here the targeting was rescued, but not the cell number. These findings gave strong indications that Psidin has two independent functions during ORN targeting and development. Psidin is predicted to be the non-catalytic part of the N-acteyltransferase complex B (NatB) in Drosophila (Brennan et al., 2007). Here, Psidin (non-catalytic) forms the NatB-complex together with dNAA20 (catalytic). This complex is thought to acetylate nascent protein chains N-terminally. In this study I demonstrated for the first time that both proteins interact in vivo and in vitro. Indicating that the NatB-complex is involved in ORN survival, the knock-down of dNAA20 in psidinIG978 mutants led to a reduction of ORN cell number that is reminiscent of the cell number in psidin1 or psidin55D4 background. At the same time, the knock-down of dNAA20 had no effect on the targeting of ORNs. Furthermore I was able to show that wild type Psidin and PsidinIG978 interact with dNAA20 at comparable levels in vitro. This is in agreement with the finding that the psidinIG978 allele selectively affects ORN targeting, but not ORN survival. In addition, I was able to map the interaction domain between Psidin and dNAA20. This revealed that the point mutation found in psidinIG978 is just outside of the minimal interaction domain. Deletion of the entire interaction domain led to a complete abolishment of the Psidin/dNAA20 interaction. Furthermore I was able to demonstrate that the interaction of Psidin and dNAA20 is regulated by the phosphorylation of a highly conserved serine residue (S678). Expression of the non-phosphorylatable Psidin isoform (S678A) rescued the targeting and cell number phenotype in vivo. Contrary expression of the phosphomimetic isoform (S678D) only rescued the targeting phenotype, but failed to restore ORN cell number in vivo. In line with this observation is the finding that the S678D isoform is unable to bind dNAA20 in vitro. At the same time the S678A isoform binds dNAA20 at normal levels in vitro. Taken together, the data presented in this work demonstrate that Psidin has two functions during the development and targeting of ORNs using two independent molecular mechanisms: First, during axon targeting Psidin is required as an actin destabilizing molecule and antagonist of Tropomyosin. Psidin maintains the lamellipodia size in growth cones and keeps the cytoskeleton in a dynamic and responsive state. This ensures that growing axons can respond properly to various guidance cues. Second, to ensure ORN survival, Psidin is required as non-catalytic part of the NatB-complex. Here, Psidin interacts with the catalytic subunit dNAA20. The formation of the NatB-complex is regulated by phosphorylation of a conserved serine. In its unphosphorylated state Psidin binds dNAA20 and ensures ORN survival, whereas phosphorylation causes the abolishment of this interaction which results in a reduction of ORN cell number. Concluding, this thesis unambiguously shows that Psidin is required at different time points during the formation of the olfactory system of Drosophila. It utilizes two different pathways to ensure (i) ORN survival as part of the NatB-complex and (ii) ORN targeting as actin binding protein. Due to its strong conservation in higher organisms, the here presented data provide important insights into the function of Psidin’s mammalian homologues.

Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored

The task of the visual system is to extract behaviourally relevant information from the visual scene. A common strategy for most animals ranging from insects to humans is to constantly reposition gaze by making saccades within the scene. This ‘fixate and saccade’ strategy seems to pose a challenge, as it introduces a highly blurred image on the retina during a saccade, but at the same time acquires a ‘snapshot’ of the world during every fixation. The visual signals on the retina are thus segmented into brief image fixations separated by global motion. What is the response of a ganglion cell to ‘motion blur’ caused by a saccade, and how does it influence the response to subsequent fixations? Also, how does the global motion signal influence the response dynamics of a ganglion cell? In this thesis, we addressed these questions by two complementary approaches. First, we analysed the retinal ganglion cell responses to simulated saccades. We analysed two important aspects of the response - 1) response during a saccade-like motion, 2) response to fixation images. For about half of the recorded cells, we found strong spiking activity during the saccade. This supports the idea that the retina actively encodes the saccade and may signal the abrupt scene change to downstream brain areas. Furthermore, we characterized the responses to the newly fixated image. While there appears to be only little influence of the preceding motion signal itself on these responses, the responses depended strongly on the image content during the fixation period prior to the saccade. Thus, saccadic vision may provide ‘temporal context’ to each fixation, and ganglion cells encode image transitions rather than currently fixated images. Based on this perspective, we classified retinal ganglion cells into five response types, suggesting that the retina encodes at least five parallel channels of information under saccadic visual stimulation. The five response types identified in this study are as follows: 1) Classical Encoders - Response only to preferred stimuli; 2) Offset Detectors - Response only to the saccade; 3) Indifferent Encoders - Response to all fixated images; 4) Change Detectors - Response only when the new image after the saccade differs from the previous image; 5) Similarity Detectors - Response only when the new image after the saccade is similar to the previous image. Second, we analysed the influence of global motion signals on the response of a retinal ganglion cell to the stimulus in its receptive field. The stimulus beyond the receptive field is designated as remote stimulus. We chose simple stimulus that represent various configurations used in earlier studies, thus allowing us to compare our results. We show that the remote stimulus both enhances and suppresses the mean firing rate, but only suppresses the evoked activity. Furthermore, we show that the remote stimulus decreases the contrast sensitivity and modifies the response gain. Thus, the ganglion cells encode the stimulus in relation to the whole scene, rather than purely respond to the stimulus in the receptive field. Our results suggest that the global motion signals provide ‘spatial context’ to the response of the stimulus within the receptive field.