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

Author

Listed:
  • Rosario Vicidomini

    (NIH)

  • Saumitra Dey Choudhury

    (NIH
    New Delhi)

  • Tae Hee Han

    (NIH)

  • Tho Huu Nguyen

    (NIH)

  • Peter Nguyen

    (NIH)

  • Felipe Opazo

    (University Medical Center Göttingen
    NanoTag Biotechnologies GmbH)

  • Mihaela Serpe

    (NIH)

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.

Suggested Citation

  • Rosario Vicidomini & Saumitra Dey Choudhury & Tae Hee Han & Tho Huu Nguyen & Peter Nguyen & Felipe Opazo & Mihaela Serpe, 2024. "Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50462-2
    DOI: 10.1038/s41467-024-50462-2
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    References listed on IDEAS

    as
    1. Thomas C. Südhof, 2008. "Neuroligins and neurexins link synaptic function to cognitive disease," Nature, Nature, vol. 455(7215), pages 903-911, October.
    2. Karzan Muhammad & Suneel Reddy-Alla & Jan H Driller & Dietmar Schreiner & Ulises Rey & Mathias A. Böhme & Christina Hollmann & Niraja Ramesh & Harald Depner & Janine Lützkendorf & Tanja Matkovic & Tor, 2015. "Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function," Nature Communications, Nature, vol. 6(1), pages 1-15, December.
    3. Hansjörg Götzke & Markus Kilisch & Markel Martínez-Carranza & Shama Sograte-Idrissi & Abirami Rajavel & Thomas Schlichthaerle & Niklas Engels & Ralf Jungmann & Pål Stenmark & Felipe Opazo & Steffen Fr, 2019. "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    4. Olena Riabinina & Darya Task & Elizabeth Marr & Chun-Chieh Lin & Robert Alford & David A. O'Brochta & Christopher J. Potter, 2016. "Organization of olfactory centres in the malaria mosquito Anopheles gambiae," Nature Communications, Nature, vol. 7(1), pages 1-12, December.
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