De rol van Tyrosine-Fosforylering tijdens Dscam1 signalisatie

Axon guidance is the developmental process during which outgrowing neurites of cells travel long distances to reach their proper synaptic targets. Precision in wiring is essential for any function of the nervous system. Therefore, axon guidance is critical to the existence of all animals. The navigation of growing neurites occurs at their tips in a sensory-motor structure known as the growth cone. The cell membranes of growth cones are equipped with axon-guidance-receptor-molecules. These trans-membrane proteins sense the composition of the environment and respond to the information by initiating intracellular signaling cascades. Hence, axon guidance receptors represent the regulatory central elements of neural wiring. Higher order species distinguish themselves from evolutionary lower species by more complex nervous systems. The formation of complex neuronal networks calls for a specialized sub-class of guidance receptors. The larger extent of the neuronal arborization requires that neurites are capable of sensing the position of a given neurite in relation to other neurites derived from the same cell. This task is assumed by “self recognition receptors”. They guarantee that a neuron covers the biggest possible area with a given set of neurites. Unnecessary “self-crosses “are avoided by triggering repulsion between neurites of the same cell. The importance of an organizing principle based on self-recognition is emphasized by the fact that it has evolved more than once. Interestingly vertebrates and invertebrates use different combinatorial systems of surface receptors to unmistakably label a neuronal surface. Among such neuronal self-recognition molecules, Drosophila Dscam1 is the first receptor described. Therefore, Dscam1 mediated neuronal self-recognition is very well understood. The 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 Dscam1 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 into gaining insights into 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 (Dascenco and Erfurth et al., 2015; He et al., 2014a). They demonstrate that the Dscam1 receptor is indispensable for the axonal patterning of mechanosensory neurons in the ventral nerve cord. In contrast to its role in uniform dendritic patterning however, 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, such as the branched arborization of mechanosensory-neurons. Dscam1 tyrosine phosphorylation is positively regulated by Src kinases and negatively modulated by the receptor tyrosine phosphatase RPTP69D. I identified three critical tyrosine residues in the intracellular domain of Dscam1 important for the interaction with RPTP69D. We also show that Dscam1 physically interacts with RPTP69D substrate traps on both of the two RPTP69D phosphatase domains. These interactions modulate Dscam1 phosphorylation, rendering Dscam1 the first identified substrate of RPTP69D as of today. In the third part of my dissertation, I summarize the results of a combination of proteomic screens. They were aimed at unraveling the Dscam1 signaling complex and identifying tyrosine phosphorylated proteins that are regulated by Dscam1 signaling. I identified new downstream targets of the pathway. 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. Among the confirmed novel Dscam1 binding partners are the receptor Pvr, the scaffolding protein α-Spectrin and the guanine nucleotide exchange (GEF) DOCK4. Surprisingly, I also detected a link of Dscam1 to the transcriptional machinery, which I confirmed via microarrays in hemocytes. Notably, some of the novel interactors, such as the Dscam1-Pvr complex, might be of special interest, because they can be detected among the vertebrate orthologue receptors as well. Taken together, this dissertation demonstrates that Dscam1 signaling is tightly regulated at several levels: An intrinsic sensitivity threshold for self-recognition is set by regulating the number of isoforms expressed on the cell surface. In a second layer, distinct ligands activate the receptor and the phosphorylation state of the intracellular domain, affecting thereby local translation, Dscam1 endocytosis and local cytoskeletal dynamics. It will be a challenge in the future, to dissect under which circumstances and in which cellular context the different signaling complexes are formed and activated.nrpages: 315status: publishe