%0 Journal Article
%T Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo.
%A Vicidomini R
%A Choudhury SD
%A Han TH
%A Nguyen TH
%A Nguyen P
%A Opazo F
%A Serpe M
%J Nat Commun
%V 15
%N 1
%D 2024 Jul 18
%M 39025931
%F 17.694
%R 10.1038/s41467-024-50462-2
%X 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.