Identification of interaction partners of unzipped, a novel cell adhesion molecule

Abstract

The brain is unquestionably the most sophisticated organ in bodies of many organisms. Establishment of this complex architecture depends on the ability of axons to find their proper targets. On their way to their synaptic sites axons follow guidance cues, which can be repulsive or attractive. Negative and positive adhesion between these guidance cues and axon guidance molecules on the cell surface of axons determine the path on which axons elongate. While some axons (pioneer axons) are steered by cues, some others (follower axons) follow the previously formed paths. Unzipped (Uzip), a novel cell adhesion molecule is expressed in neurons and glial cells and is involved in proper targeting of Drosophila olfactory receptor neurons (ORNs). Unzipped is expressed in pioneer neurons, which are guided by glia-derived Uzip through homophilic interaction. We hypothesize that Uzip localized to pioneer neurons interacts with other adhesion molecules on follower axons through heterophilic interactions. In an attempt to prove our hypothesis and understand the mechanism of Unzipped guidance function, we aimed to identify interaction partners of Uzip. For this purpose we performed co-immunoprecipitation assays followed by Mass spectrometry analysis. Among the proteins that were identified in this analysis, four candidates were selected for more detailed analysis: Hts, dFMR1, EPS15 and Syn. Co-IP experiments performed to validate the MS results did not reveal any physical interaction between these candidates and Uzip. Additionally, analysis of the expression pattern of these four proteins revealed that in most cases these do not co-localize with Unzipped, except for Hts where more detailed analyses are needed before excluding it as an interaction partner. Furthermore, the expression pattern of a Flag-HA-tagged Uzip fusion protein was analyzed throughout Drosophila nervous system development to reveal its endogenous expression pattern.