(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Characterization of the intracellular neurexin interactome by in vivo proximity ligation suggests its involvement in presynaptic actin assembly [1]

['Marcos Schaan Profes', 'Department Of Neuroscience', 'Albert Einstein College Of Medicine', 'Bronx', 'New York', 'United States Of America', 'Araven Tiroumalechetty', 'Neel Patel', 'Stephanie S. Lauar', 'Department Of Biochemistry']

Date: 2024-02

Neurexins are highly spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream of neurexin’s largely invariant intracellular domain (ICD). Caenorhabditis elegans contains a single neurexin gene that we have previously shown is required for presynaptic assembly and stabilization. To gain insight into the signaling pathways mediating neurexin’s presynaptic functions, we employed a proximity ligation method, endogenously tagging neurexin’s intracellular domain with the promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down and mass spectrometry. We compared our experimental strain to a control strain in which neurexin, endogenously tagged with TurboID, was dispersed from presynaptic active zones by the deletion of its C-terminal PDZ-binding motif. Selection of this control strain, which differs from the experimental strain only in its synaptic localization, was critical to identifying interactions specifically occurring at synapses. Using this approach, we identified both known and novel intracellular interactors of neurexin, including active zone scaffolds, actin-binding proteins (including almost every member of the Arp2/3 complex), signaling molecules, and mediators of RNA trafficking, protein synthesis and degradation, among others. Characterization of mutants for candidate neurexin interactors revealed that they recapitulate aspects of the nrx-1(-) mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to investigate a possible role for neurexin in local actin assembly, we endogenously tagged its intracellular domain with actin depolymerizing and sequestering peptides (DeActs) and found that this led to defects in active zone assembly. Together, these results suggest neurexin’s intracellular domain may be involved in presynaptic actin-assembly, and furthermore highlight a novel approach to achieving high specificity for in vivo proteomics experiments.

Funding: PTK and MSP were funded by the Simons Foundation (SFARI pilot award) and the Mathers Foundation. SS gratefully acknowledges for financial support AFAR (Sagol Network GerOmics award), Deerfield (Xseed award), Relay Therapeutics, Merck and the Einstein-Mount Sinai Diabetes Research Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Schaan Profes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To identify proteins that interact with neurexin intracellularly, we used CRISPR gene editing to endogenously tag the neurexin intracellular domain with TurboID and confirmed that this does not affect neurexin function in vivo. Streptavidin pull-downs and mass spectrometry were used to identify biotinylated proteins. We then compared our results to 3 different negative controls: a wild-type strain (N2 Bristol) lacking any TurboID protein, a strain over-expressing cytosolic TurboID pan-neuronally, and a strain in which TurboID was endogenously tagged to NRX-1, but in which the PBM of NRX-1 had been deleted leading to a de-clustering of NRX-1 from presynaptic active zones. This “ΔPBM” negative control is thus expressed from the endogenous locus, and thus in the same cells and likely at the same levels as the experimental strain and differs only in its specific localization at synapses. By comparing our experimental strain with the 3 different control strains, we find that the ΔPBM strain is the most appropriate negative control, the former 2 being too permissive or too restrictive, respectively. Using this control, we have generated a list of potential NRX-1 interactors, including both known and novel binding partners. These include presynaptic active zone proteins as well as many proteins involved in remodeling of the actin cytoskeleton. We characterized mutants for a subset of these proteins and discovered that they recapitulate aspects of the nrx-1(-) mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to directly assess the role of actin polymerization in neurexin’s presynaptic function, we fused a bacterially derived actin-sequestering peptide Gelsolin1 (GS1) to neurexin’s ICD and found that this resulted in a pronounced reduction in active zone size.

To better understand the molecules that might mediate neurexin’s presynaptic role in synapse stabilization and maturation, we have employed the enzyme-catalyzed proximity-labeling approach TurboID [ 24 ]. This method utilizes the promiscuous biotin ligase BirA, fused to a protein of interest, to allow for biotinylation of target proteins within a radius of a few nanometers. Biotinylated proteins are pulled down with streptavidin and identified by mass spectrometry. Unlike traditional biochemical approaches, this method does not require interacting proteins to remain in complex during purification, a particular advantage when studying transmembrane proteins or looking for transient interactions. While proximity ligation methods have been extensively validated in cultured cells, their application in vivo has only recently begun to reveal important biological interactions [ 25 , 26 ].

Caenorhabditis elegans contains a single neurexin gene (nrx-1) that encodes both long and short isoforms [ 19 , 20 ]. The long isoforms of NRX-1 have been implicated in neurite outgrowth, synapse specificity, and postsynaptic organization [ 21 , 22 ], while the short isoform is sufficient for presynaptic maturation and stability [ 20 ]. Using markers for presynaptic assembly including the SV-associated protein RAB-3 and the AZ protein clarinet (CLA-1; homolog of vertebrate AZ protein Piccolo [ 23 ]), we have previously shown that C. elegans NRX-1 stabilizes nascent synapses and is required for their morphological and functional maturation [ 20 ]. However, the downstream signaling pathways responsible for these functions remain unknown.

Neurexins constitute a family of presynaptic CAMs that are highly associated with autism and schizophrenia [ 6 ], and are thought to function as central “hubs” of trans-synaptic interaction [ 7 ]. The synaptogenic activity of neurexin was initially demonstrated by showing that binding to its canonical binding partner neuroligin could induce the formation of hemi-presynapses in cultured neurons [ 8 – 10 ]. The human genome encodes 3 neurexin genes, which together can be expressed as approximately 4,000 different splice isoforms [ 11 , 12 ]. These isoforms contain a mostly invariant intracellular domain (ICD) responsible for a largely uncharacterized downstream intracellular signaling pathway: the intracellular C-terminal PDZ-binding motif (PBM) of neurexin interacts with the synaptic vesicle (SV) protein synaptotagmin as well as the scaffolding proteins Cask and Mint [ 13 – 16 ]. In addition, Drosophila neurexin has been shown to interact with the active zone (AZ) protein dSYD-1 [ 17 ] as well as the actin-binding protein spinophilin [ 18 ].

The proper formation of synaptic connections underlies our brain’s ability to form appropriate neuronal circuits, and defects in this process lead to neurodevelopmental and neuropsychiatric disorders. Synaptic cell-adhesion molecules (sCAMS) are thought to play a role in both the specificity of this process, by selecting appropriate synaptic partners [ 1 – 3 ], and in the stabilization and functional maturation of nascent synapses [ 4 , 5 ].

Results

Mutants of candidates from proteomics screen partially phenocopy neurexin mutants and have varied effects on synapse assembly/stability We focused our attention on several candidate interactors that, while not previously associated with neurexin, were predicted to be involved in cytoskeletal or cell adhesion-related pathways. Null mutants for these genes, generated by the C. elegans Deletion Mutant Consortium [30], were obtained from stock centers (see strain list in Materials and methods) and crossed to our synaptic marker strain and assessed for presynaptic assembly defects. These include frm-4, hum-4, and rig-3 (Fig 4A). Frm-4 encodes a FERM domain-containing protein predicted to be involved in actomyosin structure organization, hum-4 (heavy chain of an unconventional myosin) encodes a protein that is predicted to enable actin filament binding activity and rig-3 (neuRonal IGCAM) encodes an adhesion molecule located in axons and synapses. Compared to wild-type animals, nrx-1(-) mutants exhibit an approximately 30% reduction in the number of active zones (CLA-1 puncta), primarily within the proximal synaptic domain, as well as an increase in small, asynaptic vesicle precursors (RAB-3 puncta) in the axon commissure (Figs 4B, 4C and S3, and [20]). The frm-4(-), rig-3(-), and hum-4(-) mutants all showed a pronounced reduction in CLA-1 puncta in comparison to wild type (Fig 4B and 4C). The frm-4(-) and hum-4(-) mutants also recapitulated the increase in asynaptic RAB-3 seen in the nrx-1(-) mutant (S3B Fig). The fact that disparate candidate interactors seem to regulate distinct aspects of neurexin function suggests that neurexin may function upstream of several different pathways controlling presynaptic assembly and stability. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Validation of actin-binding proteins by mutant analysis and DeAct tagging. (A) Zoom in of Semi-Volcano plot of genes corresponding to the proteins enriched in our experimental strain (neurexin-TurboID) compared to control (neurexin-DPBM-TurboID), replotted from Fig 2C, but with selected candidate interactor genes highlighted to show their relative enrichment within the dataset. (B) Straightened images of CLA-1-GFP puncta in the DA9 synaptic domain across different genotypes. Scale bar: 4 μm. (C) Quantification of CLA-1 puncta number in the indicated genotypes. (D) Schematic depicting the insertion site of DeAct tool GS1. (E) Straightened images of CLA-1-GFP puncta in the DA9 synaptic domain across wild type, nrx-1(-) and nrx-1::DeAct(GS1) genotypes. Scale bar: 4 μm. (F) Quantification of CLA-1 puncta number and size in the indicated genotypes in E. GS1, Gelsolin segment 1. https://doi.org/10.1371/journal.pbio.3002466.g004

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002466

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.