{Reference Type}: Journal Article
{Title}: Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo.
{Author}: Vicidomini R;Choudhury SD;Han TH;Nguyen TH;Nguyen P;Opazo F;Serpe M;
{Journal}: Nat Commun
{Volume}: 15
{Issue}: 1
{Year}: 2024 Jul 18
{Factor}: 17.694
{DOI}: 10.1038/s41467-024-50462-2
{Abstract}: Neurexins are key adhesion proteins that coordinate extracellular and intracellular synaptic components. Nonetheless, the low abundance of these multidomain proteins has complicated any localization and structure-function studies. Here we combine an ALFA tag (AT)/nanobody (NbALFA) tool with classic genetics, cell biology and electrophysiology to examine the distribution and function of the Drosophila Nrx-1 in vivo. We generate full-length and ΔPDZ ALFA-tagged Nrx-1 variants and find that the PDZ binding motif is key to Nrx-1 surface expression. A PDZ binding motif provided in trans, via genetically encoded cytosolic NbALFA-PDZ chimera, fully restores the synaptic localization and function of NrxΔPDZ-AT. Using cytosolic NbALFA-mScarlet intrabody, we achieve compartment-specific detection of endogenous Nrx-1, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles. Our findings illustrate the versatility of the ALFA system and pave the way towards dissecting functional domains of complex proteins in vivo.