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

In arthropods like Drosophila, Down syndrome cell adhesion molecules (Dscam1) exhibit enormous molecular diversity. A single Dscam1 gene encodes a large superfamily of neuronal cell recognition proteins that control neuronal outgrowth and anatomy. A comparable function is exhibited by the vertebrates DSCAMs of which only few isoforms exist. However, it is largely unknown, if and how this function of Dscams affects neuronal function and the control of behavior by the nervous system. In this thesis, I employed an arsenal of genetic techniques to perturb the expression level of Dscam1 isoforms in directionally selective Lobula Plate Tangential Cells (LPTCs). LPTCs of the Vertical (VS) and the Horizontal System (HS) were chosen as a model system because of their well-documented anatomy, role in information processing and behavior. Though, only little is known about the developmental mechanisms and molecular factors controlling the morphogenesis and wiring of these cells. The central aim of my study thus is to reveal a possible role of Dscam1 in the growth and development of the complex dendrites of in particular HS cells. Furthermore, my work aims at establishing a novel model system for integrated studies on the development and function of LPTCs by genetic manipulations of Dscam1 expression. My results demonstrate that Dscam1 is expressed broadly in the fly visual system including HS-cells (immunolabeling of the conserved intracellular domain). Loss of Dscam1 function and reduced isoform diversity consistently elicited misrouting and self-crossings of neurites in LPTC dendrites. In contrast, misexpression of selected single Dscam1 isoforms caused a severe reduction in the size and branching complexity of LPTC dendrites. The dendritic gain-of-function phenotype (ectopic expression of the Dscam1 isoform 11.31.25.1) was strongly dependent on the time of onset of misexpression during development. These results demonstrate that Dscam1 contributes to the development of LPTC dendrites. This system can now be used to (A) address a possible role of Dscam1 in the function of neurons and circuitries and (B) to address the interplay of anatomy and function of LPTC dendrites. In further side projects I aimed at the development of additional genetic tools for the investigation of the role of LPTCs in behavior and for studies on the wiring of LPTCs to the presynaptic circuitry. I established a heat-shock protocol for the ablation of specified LPTCs by RicinA expression and I generated a fly line for the expression of TN-XXL (a genetically encoded calcium biosensor) in small cell clusters or individual cells. Finally, I participated in efforts to establish a virus based retrograde labeling method in Drosophila.

In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membrane. Translocation of these precursor proteins across the double envelope membrane is achieved by two multimeric complexes - the so-called TOC and TIC complexes (Translocon at the Outer envelope of Chloroplast and Translocon at the Inner envelope of Chloroplast, respectively). N-terminal transit peptides essential for import of the precursor proteins are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursor proteins would enter the chloroplast through the same, jointly acting TOC/TIC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Different combinations of TOC and TIC proteins were shown to establish different import sites in Arabidopsis thaliana with specificity for either photosynthetic proteins (the general import pathway) or non-photosynthetic „housekeeping“ proteins. Moreover, numerous non-canonical import pathways such as the import of Tic32 and AtQORH mediated by the yet unknown novel import pathway and the import via the secretory pathway were shown to exist. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. Examples of the transit sequenceless precursor proteins are the chloroplast envelope quinone oxidoreductase homologue, AtQORH and the chloroplast inner envelope protein 32, Tic32. Both proteins are imported into the inner plastid envelope membrane by a non-canonical pathway (Toc159- and Toc75-independent) and without any proteolytic cleavage. In the present study not only the import characteristic of nine tentative ‘non-canonical’ chloroplast precursor proteins but also the new interactions between these precursor proteins and the proteins at the organellar surfaces were analyzed. Moreover, a non-canonical precursor protein without the classical transit peptide, the iron superoxide dismutase (FSD1) could be identified. Biochemical crosslinking experiments revealed that FSD1 interacts with new members of the Toc159 family in pea, namely PsToc132 and PsToc120. Using deletion mutants as well as a peptide scanning approach, regions of the precursor protein, which are involved in receptor binding could be defined. These are distributed across the entire sequence; surprisingly only the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the ‘non-canonical’ targeting information and recognition by a specific receptor, this precursor protein follows a similar way across the envelope as the majority of plastid precursor proteins.

In their habitats, microorganisms are often in competition for limited nutrients. In order to succeed, many Gram-positive bacteria resort to production of peptide antibiotics. Therefore, resistance mechanisms against these compounds are essential. The first step of ensuring survival is the perception of the harmful drugs and mediation of resistance against it. In recent years, a group of ABC-transporters have been recognized as important resistance determinate against antimicrobial peptides. The expression of these transporters is generally regulated by a two-component system, which in most cases is encoded next to the transporter. Together they are described as detoxification modules. The permeases of the transporters are characterized by a large extracellular domain, while the histidine kinases lack an obvious input domain. One of the best understood examples is the BceRS-BceAB system of Bacillus subtilis, which mediates resistance against bacitracin, mersacidin and actagardine. For this system it was shown that the histidine kinase is not able to detect the substrate directly and instead has an absolute requirement for the transporter in stimulus perception. This describes a novel mode of signal transduction in which the transporter is the actual sensor and therefore regulates its own expression. To date, mechanistic details for this unique mode of signal transduction remain unknown. Several other examples have been described for transport proteins that have acquired additional sensing or regulatory functions beyond solute transport, and these have been designated trigger transporters. For these bifunctional transporters a direct protein-protein interaction with membrane-integrated or soluble components of signal transduction relays has been postulated. However, for most sensor/co-sensor pairs, conclusive proof of such an interaction is lacking, and so far little is known about the sites that might mediate contacts between the putative protein interfaces and how communication is achieved. Based on sequence and architectural similarities, we identified over 250 BceAB-like transporters in the protein database, which occurred almost exclusively in Firmicutes bacteria. To whether the regulatory interplay between the ABC transporter and the two-component system was a common theme in these antimicrobial peptide resistance modules, we carried out a phylogenetic study of these identified systems. We identified a clear coevolutionary relationship between transport permeases and histidine kinases. Furthermore, we identified conserved putative response regulator binding sites in the promoter regions of the transporter operons. Additionally, we were able to provide a tool to identify TCSs for transporters lacking a regulatory system in their genomic neighbourhood, which was based on the coclustering of histidine kinases and transporter permeases. These findings also suggested the existence of a sensory complex between BceAB-like transporters and BceS-like histidine kinases. To further investigate the signaling mechanism, we performed a random mutagenesis of the transport permease BceB with the aim to identify regions or residues within the transporter that are involved in signaling and/or resistance. With this approach we were able to identify mutations that affected either the ability for signaling or mediation of resistance. This showed a partial genetic separation of the two qualities, which could be achieved by single amino acid replacements. These results provide first insights into the signaling mechanism of the Bce system. In order to analyse the proposed communication between two-component system and ABC transporter, we further characterized their interactions by in vivo and in vitro approaches. We could demonstrate that the transporter BceAB is indeed able to interact directly with the histidine kinase. Because it was unknown how the signal perception by BceAB-type transporters occurs, we next analyzed substrate binding by the transporter permease BceB and could show direct binding of bacitracin by BceB. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the activity of the histidine kinase. In summary this thesis clearly shows the existence of a sensory complex comprised of BceRS-like two-component systems and BceAB-like ABC transporters and provides first functional insights into the mechanism of stimulus perception, signal transduction and antimicrobial resistance mechanism employed by these wide spread detoxification systems against antimicrobial peptides.

Focusing at the fly visual system I am addressing the identity and function of neurons accomplishing two fundamental processing steps required for survival of most animals: neurons of peripheral circuits underlying colour vision as well neurons of higher order circuits underlying visual memory. Colour vision is commonly assumed to rely on photoreceptors tuned to narrow spectral ranges. In the ommatidium of Drosophila, the four types of so-called inner photoreceptors express different narrow-band opsins. In contrast, the outer photoreceptors have a broadband spectral sensitivity and are thought to exclusively mediate achromatic vision. Using computational models and behavioural experiments, I here demonstrate that the broadband outer photoreceptors contribute to colour vision in Drosophila. A model of opponent processing that includes the opsin of the outer photoreceptors scores the best fit to wavelength discrimination behaviour of flies. To experimentally uncover the contribution of individual photoreceptor types, I used blind flies with disrupted phototransduction (norpA-) and rescued norpA function in genetically targeted photoreceptors and receptor combinations. Surprisingly, dichromatic flies with only broadband photoreceptors and one additional receptor type can discriminate different colours, indicating the existence of a specific output comparison of outer and inner photoreceptors. Furthermore, blocking interneurons postsynaptic to the outer photoreceptors specifically impairs colour but not intensity discrimination. These findings show that outer receptors with a complex and broad spectral sensitivity do contribute to colour vision and reveal that chromatic and achromatic circuits in the fly share common photoreceptors. Higher brain areas integrate sensory input from different modalities including vision and associate these neural representations with good or bad experiences. It is unclear, however, how distinct sensory memories are processed in the Drosophila brain. Furthermore, the neural circuit underlying colour/intensity memory in Drosophila remained so far unknown. In order to address these questions, I established appetitive and aversive visual learning assays for Drosophila. These allow contrasting appetitive and aversive visual memories using neurogenetic methods for circuit analysis. Furthermore, the visual assays are similar to the widely used olfactory learning assays and share reinforcing stimuli (sugar reward and electric shock punishment), conditioning regimes and methods for memory assessment. Thus, a direct comparison of the cellular requirements for visual and olfactory memories becomes feasible. I found that the same subsets of dopamine neurons innervating the mushroom body are necessary and sufficient for formation of both sensory memories. Furthermore, expression of D1-like Dopamine Receptor (DopR) in the mushroom body is sufficient to restore the memory defect of a DopR null mutant (dumb-). These findings and the requirement of the mushroom body for visual memory in the used assay suggest that the mushroom body is a site of convergence, where representations of different sensory modalities may undergo associative modulation.

For all animals it is highly advantageous to associate an environmental sensory stimulus with a reinforcing experience. During associative learning, the neural representation of the sensory stimulus (conditioned stimulus; CS) converges in time and location with that of the reinforcer (unconditioned stimulus; US). The CS is then affiliated with a predictive value, altering the animal’s response towards it in following exposures. In my PhD thesis I made use of olfactory aversive conditioning in Drosophila to ask where these two different stimuli are represented and how they are processed in the nervous system to allow association. In the first part of my thesis, I investigated the presentation of the odor stimulus (CS) and its underlying neuronal pathway. CS-US association is possible even when the US is presented after the physical sensory stimulus is gone ('trace conditioning'). I compared such association of temporally non-overlapping stimuli to learning of overlapping stimuli ('delay conditioning'). I found that flies associate an odor trace with electric shock reinforcement even when they were separated with a 15 s gap. Memories after trace and delay conditioning have striking similarities: both reached the same asymptotic learning level, although at different rates, and both memories have similar decay kinetics and highly correlated generalization profiles across odors. Altogether, these results point at a common odor percept which is probably kept in the nervous system throughout and following odor presentation. In search of the physiological correlate of the odor trace, we used in vivo calcium imaging to characterize the odor-evoked activity of the olfactory receptor neurons (ORNs) in the antennal lobe (in collaboration with Alja Luedke, Konstanz University). After the offset of odor presentation, ORNs showed odor-specific response patterns that lasted for a few seconds and were fundamentally different from the response patterns during odor stimulation. Weak correlation between the behavioral odor generalization profile in trace conditioning and the physiological odor similarity profiles in the antennal lobe suggest that the odor trace used for associative learning may be encoded downstream of the ORNs. In the second part of the thesis I investigated the presentation of different aversive stimuli (USs) and their underlying neuronal pathways. I established an odor-temperature conditioning assay, comparable to the commonly used odor-shock conditioning, and compared the neural pathways mediating both memory types. I described a specific sensory pathway for increased temperature as an aversive reinforcement: the thermal sensors AC neurons, expressing dTrpA1 receptors. Despite the separate sensory pathways for odor-temperature and odor-shock conditioning, both converge to one central pathway: the dopamine neurons, generally signaling reinforcement in the fly brain. Although a common population of dopamine neurons mediates both reinforcement types, the population mediating temperature reinforcement is smaller, and probably included within the population of dopamine neurons mediating shock reinforcement. I conclude that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway. Altogether, my results contribute to our understanding of aversive olfactory conditioning, demonstrating previously undescribed behavioral abilities of flies and their neuronal representations.