Multimodal imaging of the neuronal Robo1 cell receptor after stimulation with its canonical ligand Slit2

The goal of this proposal is to perform a multimodal imaging of the neuronal Robo1 cell receptor with Slit2. Slit proteins and their cognate binding partners, the cell surface Roundabout (Robo) receptors family, are one of four major neuronal developmental guidance cues pathways. The implication of Slit-Robo signalling in other fundamental biological processes also makes it an attractive therapeutic target. However, while Slit2 and Robo1 are well studied there are still open questions about how Slit binding triggers Robo signalling at the cell surface. More recently, we have recombinantly produced several Slit2 constructs, including an uncleavable version (Slit2-U), all incorporating a 6xHis tag. We propose to screen these to identify the minimal version of Slit2 necessary for Robo1 endocytosis, and the cellular effect of a Slit2-U. Next, we would like to determine if Slit2 is internalised with Robo1, and if so, its cellular location and correlate this with Robo1 light microscopy.