Der Einfluss von Tyrosin-Phosphorylierung auf den Dscam1-Signalweg

The Drosophila Dscam1 gene can be spliced into thousands of different isoforms, providing the basis for a cell surface code: Each cell expresses a distinct subset of 10-50 isoforms, rendering its surface uniquely recognizable. The importance of Dscam1 for axonal and dendritic patterning has been demonstrated in numerous in vivo assays. However, surprisingly little is known regarding the signaling pathway of the Dscam1 receptor. This dissertation describes my efforts to understand the molecular mechanisms of neuronal self-recognition. My dissertation is divided into three chapters: The first two chapters consist of two published papers to which I have contributed during my time in the neuronal wiring laboratory. They demonstrate that the Dscam1 receptor is indispensable for the axonal patterning of mechanosensory neurons in the ventral nerve cord of the fly. In contrast to its role in uniform dendritic patterning, it is critical to regulate Dscam1 signaling in some sub-compartments of the outgrowing axons. Such spatial regulation of Dscam1 signaling by the novel ligand Slit and tyrosine-phosphorylation allows the formation of complicated neurite patterns. Dscam1 tyrosine phosphorylation is negatively modulated by the receptor tyrosine phosphatase RPTP69D. In chapter 3, I summarize the results of a combination of proteomic screens. They were aimed at unraveling the Dscam1 signaling complex and at identifying tyrosine phosphorylated proteins that are regulated by Dscam1 signaling. These results link the Dscam1 receptor directly to the actin and tubulin cytoskeleton and suggest that the receptor is capable of physically recruiting components of the translational machinery to the membrane. Furthermore, I found the cytoplasmic domain to be associated with components of the cellular endomembrane system, suggesting that receptor internalization might be an important regulatory mode, fine-tuning the signaling response.Das Drosophila Dscam1-Gen kann in Tausende Isoformen translatiert werden. Diese stellen die Grundlage für einen Zelloberflächencode dar: Jede Zelle exprimiert eine individuelle Kombination von 10-50 Isoformen. Dies verleiht der Zelloberfläche eine einzigartige Identität. Der Belang von Dscam1 für die Bildung von Neuriten-Verzweigungen ist in vivo überzeugend demonstriert worden. Allerdings ist wenig über den Dscam1-Signalweg bekannt. Diese Dissertation dokumentiert meine Bemühungen, die molekularen Mechanismen der neuronalen Selbsterkennung zu verstehen. Meine Dissertation gliedert sich in drei Teile: Die ersten beiden Teile umfassen zwei bereits publizierte Studien, zu denen ich als Autorin beigetragen habe. Diese zeigen, dass Dscam1 unentbehrlich für die Bildung axonaler Verzweigungen von mechanosensorischen Neuronen in der ventralen Nervenschnur der Fliege ist. Im Gegensatz zur Bildung gleichmäßiger dendritischer Muster ist es wichtig, das Dscam1-Signal in Unterregionen des auswachsendenden Axons zu regulieren. Nur wenn solch eine lokale Kontrolle des Dscam1 Signalweges durch den neu identifizierten Liganden Slit und Tyrosin-Phosphorylierung gewährleistet ist, können sich komplexe axonale Verzweigungen bilden. Die Phosphorylierung des Dscam1-Rezeptors wird durch die Rezeptor-Tyrosin-Phosphatase RPTP69D negativ reguliert. Der dritte Teil meiner Dissertation befasst sich mich mit proteomischen Experimenten. Sie waren darauf ausgerichtet Proteine zu identifizieren, die auf ein Dscam1-Signal mit der Veränderung ihres Phosphorylierungs-Status reagieren. Aus den Ergebnissen lässt sich eine direkte Verbindung zwischen Dscam1 und dem Zytoskelett ableiten. Sie legen des weiteren nahe, dass der Rezeptor auch Komponenten der Translationsmaschinerie an die Membran rekrutiert. Die Assoziation von Dscam1 mit Vesikelkomponenten legt auch nahe, dass Endozytose ein wichtiger Modus der feinabgestimmten Signalregulierung sein könnte

Netrin Signaling Breaks the Equivalence between Two Identified Zebrafish Motoneurons Revealing a New Role of Intermediate Targets

We previously showed that equivalence between two identified zebrafish motoneurons is broken by interactions with identified muscle fibers that act as an intermediate target for the axons of these motoneurons. Here we investigate the molecular basis of the signaling interaction between the intermediate target and the motoneurons.We provide evidence that Netrin 1a is an intermediate target-derived signal that causes two equivalent motoneurons to adopt distinct fates. We show that although these two motoneurons express the same Netrin receptors, their axons respond differently to Netrin 1a encountered at the intermediate target. Furthermore, we demonstrate that when Netrin 1a is knocked down, more distal intermediate targets that express other Netrins can also function to break equivalence between these motoneurons.Our results suggest a new role for intermediate targets in breaking neuronal equivalence. The data we present reveal that signals encountered during axon pathfinding can cause equivalent neurons to adopt distinct fates. Such signals may be key in diversifying a neuronal population and leading to correct circuit formation

Axonal Endoplasmic Reticulum Dynamics and Its Roles in Neurodegeneration.

The physical continuity of axons over long cellular distances poses challenges for their maintenance. One organelle that faces this challenge is endoplasmic reticulum (ER); unlike other intracellular organelles, this forms a physically continuous network throughout the cell, with a single membrane and a single lumen. In axons, ER is mainly smooth, forming a tubular network with occasional sheets or cisternae and low amounts of rough ER. It has many potential roles: lipid biosynthesis, glucose homeostasis, a Ca2+ store, protein export, and contacting and regulating other organelles. This tubular network structure is determined by ER-shaping proteins, mutations in some of which are causative for neurodegenerative disorders such as hereditary spastic paraplegia (HSP). While axonal ER shares many features with the tubular ER network in other contexts, these features must be adapted to the long and narrow dimensions of axons. ER appears to be physically continuous throughout axons, over distances that are enormous on a subcellular scale. It is therefore a potential channel for long-distance or regional communication within neurons, independent of action potentials or physical transport of cargos, but involving its physiological roles such as Ca2+ or organelle homeostasis. Despite its apparent stability, axonal ER is highly dynamic, showing features like anterograde and retrograde transport, potentially reflecting continuous fusion and breakage of the network. Here we discuss the transport processes that must contribute to this dynamic behavior of ER. We also discuss the model that these processes underpin a homeostatic process that ensures both enough ER to maintain continuity of the network and repair breaks in it, but not too much ER that might disrupt local cellular physiology. Finally, we discuss how failure of ER organization in axons could lead to axon degenerative diseases, and how a requirement for ER continuity could make distal axons most susceptible to degeneration in conditions that disrupt ER continuity

Gain-of-function mutations in the ALS8 causative gene VAPB have detrimental effects on neurons and muscles

Summary Amyotrophic Lateral Sclerosis (ALS) is a motor neuron degenerative disease characterized by a progressive, and ultimately fatal, muscle paralysis. The human VAMP-Associated Protein B (hVAPB) is the causative gene of ALS type 8. Previous studies have shown that a loss-of-function mechanism is responsible for VAPB-induced ALS. Recently, a novel mutation in hVAPB (V234I) has been identified but its pathogenic potential has not been assessed. We found that neuronal expression of the V234I mutant allele in Drosophila (DVAP-V260I) induces defects in synaptic structure and microtubule architecture that are opposite to those associated with DVAP mutants and transgenic expression of other ALS-linked alleles. Expression of DVAP-V260I also induces aggregate formation, reduced viability, wing postural defects, abnormal locomotion behavior, nuclear abnormalities, neurodegeneration and upregulation of the heat-shock-mediated stress response. Similar, albeit milder, phenotypes are associated with the overexpression of the wild-type protein. These data show that overexpressing the wild-type DVAP is sufficient to induce the disease and that DVAP-V260I is a pathogenic allele with increased wild-type activity. We propose that a combination of gain- and loss-of-function mechanisms is responsible for VAPB-induced ALS

Transcriptional profiling of Drosophila larval ventral nervous system hemilineages

Over 90% of neurons in the adult CNS of Drosophila are born from neuronal stem cells (neuroblasts) during the post-embryonic phase of neurogenesis. Most of the post-embryonic neurons derive from type I neuroblasts, which undergo repeated asymmetric divisions to produce a series of ganglion mother cells (GMCs). Each GMC then divides once resulting in two neurons, the “A” (Notch-on) and “B” (Notch-off) daughters. The respective daughter neurons of each type then constitute the A and B hemilineages for that neuroblast. 33 postembryonic hemilineages contribute neurons to each thoracic hemisegment, and these immature neurons arrest their development at a similar stage until metamorphosis. These arrested neuroblast lineages are uniquely identifiable by morphology. Access to a large pool of clonally-related and morphologically similar neurons makes this system tractable to RNA-seq analysis, since one can genetically label and isolate many cells per animal, which are predicted to share similar gene expression profiles. Our primary focus is to examine hemilineages with similar targets (e.g. leg neuropil) to identify genes that are required to establish and maintain hemilineage identity early in development. Given that activating these hemilineage neurons as a group drives distinct behaviors and that they form morphologically coherent structural units during development, we hypothesized that these hemilineages should express patterns of genes that are: 1) distinct from other hemilineages and 2) characteristic of individual hemilineages. We have used hemilineage-specific GAL4 lines to isolate hemilineages for RNA-seq analysis, ultimately gathering data for 11 of the 33 hemilineages as well as for some larger populations of neurons. We found that, in addition to combinatorial patterns of genes specifying the hemilineage neurons, there are some genes that are expressed by only a single hemilineage within the ventral nervous system (VNS). Most hemilineages display unique expression of certain transcription factors (TFs) and axon guidance genes. We collected data for two pairs of sibling hemilineages (lineage 1 and lineage 12) in order to identify differences between the A and B hemilineages derived from a common neuroblast. While A neurons display greater overall transcriptional diversity than B neurons, sibling hemilineages share very similar expression profiles. Comparing the gene expression between immature and mature larval neurons revealed that mature neurons express many genes not expressed in immature neurons, such as neuropeptide signaling genes and many neurotransmitter and ion channel genes associated with mature neuron function. Birth order also appears to dictate many differences in expression profile. Late-born immature neurons are typified by a period of transient Notch-related gene expression that is absent from early-born neurons. We are characterizing the function of many differentially expressed genes in particular hemilineages.All funding was provided by the Howard Hughes Medical Institute

Molecular mechanisms underlying presynaptic plasticity: characterization of the RIM1α and SV2A interactome

Synaptic plasticity encompasses various cellular mechanisms, which confer synapses the ability to react and adapt to ongoing changes in network activity. Some of the suggested mechanisms include remodelling and/or assembly of active zones (AZ), and modulation of neurotransmitter release. At the molecular level posttranslational modifications of proteins, e.g. phosphorylation, have been reported to be associated with these events. Two components of the release machinery, RIM1α and synaptic vesicle protein 2A (SV2A) were shown to be actively involved in presynaptic plasticity. However, the impact of posttranslational modifications, like phosphorylation, on the function of these proteins is not well understood. Therefore, the goals of this thesis were to examine the impact of phosphorylation on the binding properties of RIM1α and to identify and analyse novel binding partners for SV2A. We found that the distribution of RIM1α at synapses is altered after globally increasing the level of phosphorylation, while the total level remained unchanged, suggesting that the association of RIM1α with the CAZ is controlled by its phosphorylation status. Affinity purification and MS revealed that alterations in the phosphorylation status of RIM1α affected its affinity to specific binding partners. Out of the identified proteins, four candidates with a potential functional link were chosen to be further analysed in binding assays: two kinases (unc-51-like kinase 1/2, serine arginine protein kinase 2), one calcium-binding protein (Copine VI), and proteins involved in trafficking (vesicle-associated membrane protein (VAMP) associated-protein A/B). Interestingly, RIM1α may represent the first AZ substrate for ULKs and SRPK2, which in D.melanogaster have already been linked to the assembly of AZs. This may support the hypothesis that both ULKs and SRPK2 could be actively involved in controlling not only RIM1α’s function but also its association with the CAZ. VAP proteins, by specifically binding the C2A-domain of RIM1α, may contribute to control the trafficking of RIM1α to the synapse. Copine VI may regulate the function of RIM1α in a calcium-dependent manner. Further analysis will reveal if these novel interactions may have any functional relevance for the function of RIM1α. The last part of the study was dedicated to another presynaptic protein, SV2A. To date the role played by SV2A in SV priming is not fully elucidated. Therefore, to gain insight into the enigmatic function of SV2A identification of novel binding partners was pursued. Different affinity purification strategies coupled to MS were performed in order to identify the SV2A proteome. However, none of these approaches resulted in the identification of novel interacting proteins, which could be further verified in biochemical assays. Taken together, the findings of this thesis may form the basis for further functional studies in order to decipher the molecular mechanisms underlying the function of RIM1α and in consequence, the role of RIM1α in presynaptic plasticity

An RNAi-mediated genetic screen identifies genes that promote tumour progression in a living epithelium

The complex process by which cancer cells invade local tissue and metastasise is responsible for approximately 90% of cancer related deaths. The cell biological events that underlie this transition to malignancy are driven by invariable alterations within the genome, however relatively little is known about the genetic determinants involved. If identified, novel genes which perturb the rate of tumour progression could become potential targets for future therapeutic intervention. Using a novel in vivo system, it is possible to characterise the behaviour of transformed cells during the early stages of tumour development and follow these cells in real time, thus improving our understanding of the critical events that initiate cell proliferation, tumour cell invasion and metastasis. Using Drosophila as a model organism it is possible to generate neoplastic tumours within the dorsal thorax whereby clones of transformed cells are homozygous mutant for a specific tumour suppressor gene. By specifically labelling these transformed cells with GFP, their behaviour can be observed in high temporal and spatial resolution within the living epithelium. RNAi technology can also be employed to simultaneously knock-down expression of an additional gene specifically within the mutant tissue. This forms the basis of a large-scale screen for novel genes that may promote tumour progression in this epithelium. The screen is now almost complete and so far we have screened through almost 500 genes, the majority of which have previously been implicated in cancer but remain uncharacterised. We have observed a wide range of phenotypes, with genes affecting cell proliferation, invasion, cell shape, actin organisation, junction integrity and epithelial multilayering. By setting ‘thresholds’ for particular phenotypes ‘hits’ have been identified which drastically enhance tumour progression, and these genes are in the process of being fully characterised to further our understanding of their role in tumour progression