Developing New Methods to Image and Reconstitute Complex Proteins

The top diagram is a line with segments labeled yellow, purple, gray, and green. The microscopy images are in two rows with four panels each, showing segments of spinal cord against a black background.

Reconstitution of Nrx-1 using the “split” ALFA system. The top diagram shows the complex topology of Nrx-1-DPBM-AT, the reconstitution strategy, the ALFA tag (AT), and the Nanobody-PDZ binding motif (PBM) chimera. The confocal microscopy images show larval ventral (“spinal”) cords labeled for AT (green) and Nrx-1 (magenta). In the absence of PBM, truncated Nrx-1 is retained in the endoplasmic reticulum. The addition of PBM can restore the normal distribution of reconstituted Nrx-1 (NrxDPBM-AT+ Nb-PBM). Scale bars: 10 µm.
Credit: Serpe Lab, NICHD/NIH

Neurexins are adhesion proteins critical for the formation of synapses, which connect neurons and help transmit signals. They are multidomain proteins and are found in relatively small quantities, making them difficult to study. Mutations in genes encoding neurexins are implicated in various neurodevelopmental and neuropsychiatric disorders, including schizophrenia and autism spectrum disorder.

  • In a study from the Serpe Lab, researchers developed a new system to study Neurexin-1 (Nrx-1) in vivo using the fruit fly model. The new tool will enable scientists to better understand the location and function of neurexins or other synaptic proteins and how they may contribute to human disorders.
  • The team used an ALFA tag/nanobody system and combined it with classic genetics, cell biology, and electrophysiology to examine the distribution and function of ALFA tagged-Nrx-1 in vivo.
  • The researchers made key discoveries about Nrx-1. For example, they found that the PDZ binding motif (PBM) is key to Nrx-1 surface delivery. They restored the location and function of truncated Nrx-1 by returning the PBM with a nanobody-PBM chimera.
  • The new system enabled the study team to detect Nrx-1 in various areas, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles.
  • Overall, the findings illustrate the versatility of the ALFA system and pave the way towards studying functional domains of other complex proteins in vivo.

Reference

Vicidomini R, Choudhury SD, Han TH, Nguyen TH, Nguyen P, Opazo F, and Serpe M. Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo. Nature Communications DOI: 10.1038/s41467-024-50462-2 (2024)

Learn more about the Cell Regulation and Development group: https://www.nichd.nih.gov/about/org/dir/affinity-groups/CRD.

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