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.2014 Nov 5;34(45):15083-96.
doi: 10.1523/JNEUROSCI.0348-14.2014.

Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation

Affiliations

Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation

Theodoros Tsetsenis et al. J Neurosci..

Abstract

Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

Keywords: SynView; microscopy; neurexin; neuroligin; split-GFP; synapses.

Copyright © 2014 the authors 0270-6474/14/3415083-14$15.00/0.

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Figures

Figure 1.
Figure 1.
Comparison of different approaches to visualizing synapses with neurexins and neuroligins and the split-GFP systems.A, OriginalC. elegans GRASP (Feinberg et al., 2008) in which the split-GFP moieties are fused to the N-termini ofC. elegans neuroligin-1 (ceNL1; accession no. Q9XTG1).B, Transgenic GRASP (Yamagata and Sanes, 2012) in which the split-GFP moieties were fused to the N-termini of rat neuroligin-1 (rNL1; accession no. Q62765) and rat neurexin-1β (rNrx1β; accession no. Q63373).C, mGRASP (Kim et al., 2012) in which the split-GFP moieties are fused to the C-terminal sequences of mouse neuroligin-1 (mNL1; accession no. Q99K10) and to the N-terminus of human CD4-2 (hCD4-2; accession no. P01730; residues in blue) that in turn was fused to the C-terminal sequences of rat neurexin-1β (residues in red).D, SynView (this study) in which the split-GFP moieties were inserted into rat neuroligin-1 and neurexin-1β at positions predicted from the crystal structure of their complex to place them into close proximity. The proteins are drawn approximately to scale for comparison purposes; numbers indicate residue numbers. GFP1–10 and GFP11 denote the split-GFP moieties containing β-strands 1–10 and 11, respectively. SP, Signal peptide; EHD, Esterase homology domain; and LNS, laminin/neurexin/sex hormone binding domain.
Figure 2.
Figure 2.
Design and validation of SynView1.A, Constructs used for the development of the SynView1 system. Numbers represent amino acid residues in sequences of rat neuroligin-1 (NL1) lacking splice site B (accession no. Q62765) and rat neurexin-1β (Nrx1β) lacking splice site 4 (accession no. Q63373). For the neurexin-binding mutant NL1mt32-GFP11S2, 5 point mutations were incorporated into the original NL1-GFP11S2 construct (L399A/N400A/D402N/E297A/K306A; Ko et al., 2009). SP, Signal peptide; F, Flag epitope; EHD, Esterase homology domain; TM, transmembrane region; CT, cytoplasmic tail; and LNS, laminin/neurexin/sex hormone binding domain.B, Graphic representation of the predicted structure of Nrx1β-GFP1–10S2 bound to NL1-GFP11S2, allowing the reconstitution of the two split-GFP fragments.C, HEK293 cells expressing different SynView1 constructs as indicated were mixed and the reconstituted GFP fluorescence was imaged after a 24 h coincubation in suspension culture. For EGTA-treated cells (fourth panel), cells were incubated a further 24 h in 5 mm EGTA. Scale bars, 10 μm.D, HEK293 cells expressing Nrx1β-GFP1–10S2Flag plus cerulean were mixed with cells coexpressing NL1-GFP11S2Flag plus tdTomato; development of GFP signal in the border of cell membrane contacts was monitored by fluorescence microscopy using three-color imaging for cerulean (blue), tdTomato (red), and GFP (green) Scale bar, 5 μm.E, HEK293 cells expressing Nrx1β-GFP1–10S2 were mixed with cells expressing NL1-GFP11S2Flag or NL1mt32-GFP11S2Flag and nonpermeabilized cells were immunostained with an anti-Flag antibody (blue) to visualize surface-exposed NL1-GFP11S2Flag or NL1mt32-GFP11S2Flag and an anti-GFP antibody (red) to label surface-exposed Nrx1β-GFP1–10S2. Cells were then imaged in a confocal microscope (green, SynView1 GFP-fluorescence); note that the GFP fragment present in Nrx1β-GFP1–10S2 reacts with GFP antibodies but does not exhibit fluorescence on its own and that, even though in the fifth panel, cells expressing Nrx1β-GFP1–10S2 and NL1mt32-GFP11S2Flag are brought into close proximity, no GFP reconstitution occurs. Scale bars, 5 μm.F, HEK293 cells expressing Nrx1β-GFP1–10S2Flag were mixed with cells expressing NL1-GFP11S2Flag and the reconstituted GFP fluorescence was imaged at the indicated time points after coincubation in suspension culture. Scale bars, 5 μm. Data show representative experiments independently repeated at least 3 times.
Figure 3.
Figure 3.
Binding affinities of Neurxin-1β and Neuroligin-1 are modestly increased upon insertion of split-GFP components.A, Purified soluble Ig-Nrx1β and Nrx1β-GFP1–10S2 fusion proteins containing the entire extracellular neurexin-1β sequences were analyzed by SDS-PAGE and Coomassie staining. HC, Heavy-chain Ig.B, Mock-transfected HEK293 cells or HEK293 cells expressing neuroligin-1 (NL1) or NL1-GFP11S2Flag were incubated with increasing concentrations of soluble Ig-Nrx1β or Ig-Nrx1β-GFP1–10S2 proteins, washed, and reacted with an HRP-conjugated antibody against the Ig domain. Ig-fusion-protein binding was determined colorimetrically and plotted as a function of Ig-Nrx1β or Nrx1β-GFP1–10S2 concentration after subtraction of the amount bound on mock-transfected cells. Data show representative experiment independently repeated multiple times.C, Binding affinities for each pair of neurexin-1β- and neuroligin-1-binding partners were calculated by Scatchard analysis. Affinities are shown as meanKd values ± SEM (Nrx1β,n = 4; Nrx1β-GFP1–10S2,n = 3).
Figure 4.
Figure 4.
Analysis of artificial synapse formation between neurexin-1β expressing hippocampal neurons and neuroligin-1 expressing COS-7 cells using SynView1.A–D, Cultured hippocampal neurons were infected with a lentivirus expressing Nrx1β-GFP1–10S2 and cocultured with COS-7 cells expressing NL1-GFP11S2Flag (A,C), wild-type NL1 (NL1Flag;B) or NL1mt32-GFP11S2Flag (which lacks neurexin-binding;D). Reconstituted GFP fluorescence (green) representing SynView signal was combined with two-color immunoflurorescence labeling using antibodies against the Flag epitope to detect wild-type NL1Flag, NL1-GFP11S2Flag and NL1mt32 GFP11S2Flag (blue) (A–D) against GFP to detect Nrx1β-GFP1–10S2 (the split-GFP component of which reacts with GFP antibodies; red,A), or against synapsin to mark presynaptic terminals (red,B–D). Data show representative experiments independently repeated at least 3 times. Scale bars, 10 μm.E, Quantitative analysis of synapsin puncta intensities on COS-7 cells transfected with different NL1 constructs after 48 h of coculture with hippocampal neurons expressing Nrx1β-GFP1–10S2 (data are means ± SEM and are normalized to the respective NL1 signal;n = 3 independent experiments).
Figure 5.
Figure 5.
Time course of GFP complementation during artificial synapse formation monitored by SynView1.A, Representative images of COS-7 cells transfected with NL1-GFP11S2Flag and cocultured for 5, 8, 12, 24, and 48 h with hippocampal neurons expressing Nrx1β-GFP1–10S2. Cultured neurons were fixed and stained with antibodies to the Flag epitope of NL1-GFP11S2Flag (blue) and to the synaptic vesicle protein synapsin (red) and simultaneously imaged for native GFP fluorescence from the SynView1 signal. Scale bar, 5 μm. Data show representative experiments independently repeated at least 3 times.B, Quantitative analysis of the SynView1 and synapsin puncta intensities developing as a function of time on COS-7 cells during artificial synapse formation assays (data are means ± SEM and are normalized to the NL1-GFP11S2Flag signal;n = 3 independent experiments with minimum 15 cells at each time point).
Figure 6.
Figure 6.
Visualization of synapses using SynView1 in cultured hippocampal neurons.A, Representative image of a transfected hippocampal neuron expressing NL1-GFP11S2 that is surrounded by neurons expressing Nrx1β-GFP1–10S2. Punctate SynView1 GFP fluorescence (green) was combined with immunofluorescence staining for NL1-GFP11S2 (using an anti-HA antibody; red) and synapsin (blue). Scale bar, 10 μm.B, High-magnification image of the boxed area inA showing individual and composite images of the Synview signal and NL1-GFP11S2 and synapsin-positive puncta. Scale bar, 10 μm.C,D, Quantitative analysis of the fraction of SynView1-positive (C) and NL1-GFP11S2-positive puncta (D) that are adjacent to synapsin-positive puncta. Data represent means ± SEM;n = 3 independent experiments.E, Representative image of a transfected hippocampal neuron expressing NL1mt32-GFP11S2 that is surrounded by neurons expressing Nrx1β-GFP1–10S2. No GFP fluorescence was observed, indicating inability of reconstitution of the split-GFP moieties. Cultures were stained for NL1mt32-GFP11S2 (using an anti-HA antibody; red) and synapsin (blue). Scale bar, 10 μm.F, High-magnification images of the boxed area inE showing individual and composite images of the different stainings. Scale bar, 10 μm.G, Quantitative analysis of the fraction of NL1mt32-GFP11S2-positive puncta that are adjacent to synapsin-positive puncta. Data represent means ± SEM;n = 3 independent experiments. Data show representative experiments independently repeated at least 3 times.
Figure 7.
Figure 7.
Analysis of SynView1-positive synapses in cultured neurons.A, Representative image of a transfected hippocampal neuron expressing NL1-GFP11S2 that is surrounded by neurons expressing Nrx1β-GFP1–10S2. Punctate SynView1 GFP fluorescence (green) was combined with immunofluorescence staining for vGAT (red) and vGLUT-1 (blue). Scale bar, 10 μm.B, High-magnification images of the boxed dendritic segment inA showing individual and composite images of the different stainings. Scale bar, 10 μm.C, Quantitative analysis of the percentage of SynView1-positive puncta that are adjacent to vGLUT1- or vGAT-positive puncta. Data represent means ± SEM;n = 3 independent experiments; **p < 0.01. Data show representative experiments independently repeated at least 3 times.
Figure 8.
Figure 8.
Development of neuroligin-2-based SynView2.A, Schematic drawing of the NL2-GFP11S2 construct used for SynView2. Numbers represent amino acid residues of rat neuroligin-2 (accession no. Q62888). SP, signal peptide; EHD, Esterase homology domain; TM, transmembrane region; and CT, cytoplasmic tail.B, HEK293 cells expressing Nrx1β-GFP1–10S2 were mixed with cells expressing NL2-GFP11S2 and development of the reconstituted GFP signal at the border of cell membrane contacts was monitored by fluorescence microscopy. Scale bar, 10 μm.C, Image of a hippocampal neuron sparsely transfected with NL2-GFP11S2 surrounded by neurons expressing lentivirally transduced Nrx1β-GFP1–10S2. Punctate SynView2 GFP fluorescence (green) was combined with immunofluorescence staining to NL2-GFP11S2 (using an anti-HA antibody; red) and synapsin (blue).D, High-magnification images of the boxed dendritic area inC illustrate individual and composite images of the Synview2 signal and NL2-GFP11S2 and synapsin-positive puncta. Scale bar, 10 μm.E,F, Quantitative analysis of the fraction of SynView2-positive (E) and NL1-GFP11S2-positive puncta (F) that are adjacent to synapsin-positive puncta. Data represent means ± SEM;n = 3 independent experiments. Data show representative experiments independently repeated at least 3 times.
Figure 9.
Figure 9.
Analysis of SynView2-positive synapses in cultured neurons.A, Representative image of a transfected hippocampal neuron expressing NL2-GFP11S2 that is surrounded by neurons expressing Nrx1β-GFP1–10S2. Punctate SynView1 GFP fluorescence (green) was combined with immunofluorescence staining for vGAT (red) and vGLUT-1 (blue). Scale bar, 10 μm.B, High-magnification images of the boxed dendritic segment inA showing individual and composite images of the different stainings. Scale bar, 10 μm.C, Quantitative analysis of the percentage of SynView2-positive puncta that are adjacent to vGLUT1- or vGAT-positive puncta. Data represent means ± SEM;n = 3 independent experiments; *p < 0.05. Data show representative experiments independently repeated at least 3 times.
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