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Abstract

During neuron development, the precise wiring of neuronal network is mainly secured by molecular probes that prevent synapse formation with sister dendrites, generating repulsive signals. At the same time, cell surface receptors dictate the axon orientation and growth, in response to signaling sources (guidance cues) through attractive or repulsive mechanisms. Down Syndrome Cell Adhesion Molecule (DSCAM) is a cell surface receptor, which is, mainly, expressed in commissural axons in mammals and controls neuronal response through heterophilic binding to guidance cues (i.e. netrin-1). DSCAM:netrin-1 interaction induces axon chemoattraction that triggers cytoplasmic signaling, leading to axonal growth. In addition, DSCAM mediates homophilic interactions, essential for promoting cell adhesion and aggregation. Its homologue in D. melanogaster, has a different role and is mainly involved in neuron self-avoidance and dendritic discrimination, through homophilic binding. The aim of the thesis was to identify the domains involved in homodimerization of human DSCAM and explore its heterophilic interactions with netrin-1, in order to assess the mechanistic differences between the two species. To this end, biophysical and structural studies involving SAXS, EM and X-ray crystallography were performed. A selection of DSCAM constructs, comprising the N-terminal Immunoglobulin domains Ig1-Ig4, Ig1-Ig8 and Ig1-Ig9, was designed, expressed and isolated at high purity. Diffracting crystals were successfully grown for all DSCAM constructs. For DSCAM Ig1-Ig4 diffraction data collected at 2.35 Å resolution (space group C2221) allowed its 3D structure determination. DSCAM domains adopted a horseshoe shape arrangement (as it was also suggested by EM negative staining for DSCAM Ig1-Ig9), forming a dimer in the asymmetric unit. Dimer formation was shown to be induced by crystal packing interactions and the biological assembly of DSCAM Ig1-Ig4 seemed to be monomeric, as indicated by the results obtained for this construct in solution (SAXS and SLS coupled with FPLC). In the presence of netrin-1, DSCAM Ig1-Ig9 was predominantly depicted as a monomer by EM (negative staining); however, the dimeric population observed was probably induced by netrin-1 with the identity of the second molecule not being clearly resolved. SAXS analysis indicated that a plausible complex between DSCAM Ig1-Ig9 and netrin-1 could be formed under the conditions used in the study, but further investigation is required to confirm complex formation. Overall, the present study demonstrates that the results obtained, contribute to the preliminary characterization of human DSCAM. Although the horseshoe shape configuration is conserved among the species, the underlying mechanism of human DSCAM homophilic interactions differs from Dscam in D. melanogaster. However, this finding requires further investigation, including the 3D structure determination of DSCAM Ig1-Ig9 in order to elucidate its functional role. In addition, the applied integrated structural biology approaches determined the workflow towards understanding the structure-function relationship that dictates the interactions of DSCAM and netrin-1.