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[Preprint]. 2023 Nov 17:2023.11.17.567618.
doi: 10.1101/2023.11.17.567618.

Protein-lipid interactions drive presynaptic assembly upstream of cell adhesion molecules

Elisa B Frankel  1 , Araven Tiroumalechetty  1 , Parise S Henry  1 , Zhaoqian Su  2 , Yinghao Wu  3 , Peri T Kurshan  1   4
Affiliations

Protein-lipid interactions drive presynaptic assembly upstream of cell adhesion molecules

Elisa B Frankel et al. bioRxiv. .

Abstract

Textbook models of synaptogenesis position cell adhesion molecules such as neurexin as initiators of synapse assembly. Here we discover a mechanism for presynaptic assembly that occurs prior to neurexin recruitment, while supporting a role for neurexin in synapse maintenance. We find that the cytosolic active zone scaffold SYD-1 interacts with membrane phospholipids to promote active zone protein clustering at the plasma membrane, and subsequently recruits neurexin to stabilize those clusters. Employing molecular dynamics simulations to model intrinsic interactions between SYD-1 and lipid bilayers followed by in vivo tests of these predictions, we find that PIP2-interacting residues in SYD-1's C2 and PDZ domains are redundantly necessary for proper active zone assembly. Finally, we propose that the uncharacterized yet evolutionarily conserved short γ isoform of neurexin represents a minimal neurexin sequence that can stabilize previously assembled presynaptic clusters, potentially a core function of this critical protein.

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Conflict of interest statement

DECLARATION OF COMPETING INTERESTS The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Synapse stabilization functions of NRX-1 are mediated by its intracellular domain
1A. Schematic of C. elegans neurexin/nrx-1 gene, isoforms, mutant alleles, and protein domains. The nrx-1 gene encodes both α/long isoforms and γ/short isoforms. Protein regions encoded by the nrx-1 gene are annotated at the bottom of the schematic (L/LNS = laminin-α, neurexin, sex hormone-binding globulin domain; E/EGF = EGF-like repeats; TM = transmembrane; PBM = PDZ binding motif). The fluorescent protein Skylan-S was inserted immediately prior to the C-terminal PBM. 1B. NRX-1 protein isoform schematics. The α/long isoform of NRX-1 has an extracellular domain with protein interaction motifs (yellows). The γ/short isoform protein has a small extracellular domain with no canonical protein interaction domains. The myr::ICD allele was engineered by removing extracellular sequence and replacing the transmembrane domain of nrx-1 with a myristoylation sequence (red). 1C. Schematic of the DA9 excitatory motor neuron. The axon of DA9 forms a stereotypical set of synapses (red) in a specific position (box). 1D. DA9 presynapses (labeled by CLA-1::GFP) are reduced in nrx-1(−) animals, with puncta chiefly lost from the posterior region of DA9’s synaptic zone (indicated by a red box). DA9-specific expression of nrx-1 isoform transgenes were assessed for rescue of the nrx-1(−) loss of presynapse phenotype. Three representative maximum intensity projections of the DA9 synaptic zone are stacked together for each genotype. Scale, 5 μm. 1D. Quantification of DA9 presynaptic puncta number in rescues of nrx-1(−). Sample size (number of worms) is indicated at the base of each bar, and individual values from each worm are represented as scatter points over each bar. Bars represent mean synapse number ± SD. (*p< 0.05, **p<0.01, ****p<0.001, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test).
Figure 2.
Figure 2.. NRX-1 synaptic localization is mediated by its intracellular domain
2A. Schematic of the C. elegans body plan, with imaging regions used in this paper indicated by dashed boxes. 2B. Endogenously labeled NRX-1::Skylan and CLA-1::mScarlet in CTRL and nrx-1(ΔL) animals. Merged images show magnifications of dashed box regions with NRX-1::Skylan pseudocolored in green and CLA-1::mScarlet in magenta. Scale, 5 μm. Merge scale, 1 μm. 2C. Quantification of NRX-1::Skylan puncta number and intensity in neurexin isoform alleles. 2D. Representative maximum projection images of endogenously labeled NRX-1 isoforms in the nerve ring in the heads of L4-stage animals. The dotted line indicates the boundary of the nerve ring. Saturated spots outside the nerve ring are autofluorescent granules. Scale, 5 μm. 2E. Quantification of mean NRX-1::Skylan isoform intensity in the nerve ring. 2F. Representative images of a strain expressing endogenous CLA-1::mScarlet and endogenous myristoylated intracellular domain of NRX-1::Skylan. Scale, 5 μm. Merge scale, 1 μm.
Figure 3.
Figure 3.. Intracellular scaffold protein SYD-1 recruits neurexin to presynapses using a PDZ interaction
3A. Representative images of endogenously tagged NRX-1::Skylan and SYD-1::mScarlet in mutants that disrupt the PDZ-based interaction. Note that NRX-1 declusters in all three mutant genotypes, while SYD-1 remains competent to cluster in both the nrx-1(ΔPBM) and syd-1(ΔPDZ) backgrounds. The syd-1(−) allele does not contain CDs to express a fluorescent protein. Scale, 5 μm. Merge scale, 1 μm. 3B,C. Quantification of NRX-1::Skylan (B) or SYD-1::mScarlet (C) puncta number and intensity in control and mutant alleles of syd-1 and nrx-1. 3D. Quantification of % NRX-1 ROI area co-localized with SYD-1 in syd-1(ΔPDZ) and nrx-1(ΔPBM). 3E. CLA-1::GFP-labeled presynapses in the DA9 neuron of nrx-1(−), nrx-1(ΔPBM), syd-1(ΔPDZ), and nrx-1(ΔPBM); syd-1(ΔPDZ) mutants. Scale, 5 μm. 3F. Quantification of DA9 presynapse number in mutants that disrupt the PDZ interaction between NRX-1 and SYD-1.
Figure 4.
Figure 4.. SYD-1 accumulation precedes that of NRX-1 at nascent presynapses
4A. Embryos at different developmental timepoints expressing endogenously labeled SYD-2, SYD-1, and NRX-1. The developing nerve ring at each stage is enclosed with a box (colored red at the stages in which each protein is first detected). SYD-1 and NRX-1 were imaged together in the same animals, and single planes of interest from confocal z-stacks are displayed. Scale, 10 μm. 4B,C. Endogenously labeled SYD-1 in the embryonic nerve ring (boxed), left, and in the dorsal nerve cord of L4-stage animals (right). Embryo scale, 10 μm. L4 DNC scale, 2.5 μm. 4C. Quantification of SYD-1 puncta in a nrx-1 mutant in the L4-stage DNC.
Figure 5.
Figure 5.. MD simulations predict interactions between SYD-1 C2 and PDZ domains and PIP2 phospholipids
5A. Snapshots from an atomistic MD simulation in which an Alphafold-predicted SYD-1 C2 domain structure was tested for interaction with a theoretical membrane over the course of 200 nanoseconds. The amino acids participating in interactions with the membrane are indicated in blue, and the PIP2 phospholipids in the membrane are green. 5B-E. The minimum protein-lipid distance as a function of time is represented by a different color for each of the trajectory repeats per simulation condition, with black lines representing a rolling average distance between the domain and membrane at each time point.
Figure 6.
Figure 6.. The C2 and PDZ domains of SYD-1 redundantly mediate SYD-1 synaptic localization in vivo
6A. Representative images of endogenously labeled SYD-1 lacking its C2 or PDZ domains (or both), as well as the syd-1(ΔPDZ C2*) allele, in which the PDZ domain was removed and the predicted PIP2-interacting residues of the C2 domain were mutated to alanine. Dashed boxes indicate regions used for merged magnifications at right, which depict NRX-1 in green and SYD-1 in magenta. Scale, 5 μm. Merge scale, 1 μm. 6B. Quantifications of NRX-1 puncta number and puncta intensity in syd-1 mutant alleles. 6C. Quantification of SYD-1 puncta number and puncta intensity in syd-1 mutant alleles. 6D. Quantification of co-localization between endogenous NRX-1::Skylan and SYD-1::mScarlet in syd-1 mutant alleles. 6E. The presynaptic scaffold protein SYD-1 coordinates synapse assembly through interactions with PIP2 and subsequently recruits downstream synapse components, including neurexin. Early in synapse development, SYD-2/Liprin-α and ELKS-1 form phase-separated condensates that recruit and concentrate downstream active zone proteins, enabling them to participate in the multivalent interactions necessary to build and organize functional presynapses (panel 1). Following the expression of SYD-2 and ELKS (but prior to most other active zone proteins), SYD-1 protein becomes detectable at nascent presynapses. SYD-1 is recruited to the plasma membrane through interactions between its C2 and PDZ domains and PIP2 phospholipids (panel 2). SYD-1 may recruit the phase-separated condensate through interactions with SYD-2 and ELKS-1 (panel 2), and coordinates the recruitment of NRX-1/neurexin to presynapses through PDZ-based interactions (panel 3). The γ /short isoform of NRX-1 is the isoform predominantly expressed by the C. elegans nervous system and functions in synapse stabilization.
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