Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo

Rosario Vicidomini, Saumitra Dey Choudhury, Tae Hee Han, Tho Huu Nguyen, Peter Nguyen, Felipe Opazo and Mihaela Serpe ()
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Rosario Vicidomini: NIH
Saumitra Dey Choudhury: NIH
Tae Hee Han: NIH
Tho Huu Nguyen: NIH
Peter Nguyen: NIH
Felipe Opazo: University Medical Center Göttingen
Mihaela Serpe: NIH

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-50462-2

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