2.1.

Nanoscale Organization of Nrx1$\beta$ at Presynapses

To image Nrx at presynapses, primary rat hippocampal neurons were electroporated at the time of plating (day in vitro DIV 0) with a $\mathrm{Nrx}1\beta$ construct carrying a 15 amino acid $N$-terminal biotin acceptor peptide (AP) tag (AP-$\mathrm{Nrx}1\beta$), together with the endoplasmic reticulum-resident biotin ligase (${\mathrm{BirA}}^{\mathrm{ER}}$),¹⁷ and a VGlut1-Super Ecliptic (SEP) presynaptic reporter. Biotin naturally present in the culture medium is covalently added to a central lysine residue in the AP-tag through the enzymatic action of the biotin ligase, and AP-$\mathrm{Nrx}1\beta$ reaches the plasma membrane in a biotinylated form.²⁵ The biotinylated tag is then detected by incubation with monomeric streptavidin (mSA) conjugated to an organic dye (Alexa647), compatible with dSTORM. In DIV 15 neurons, we observed an excellent colocalization of the Alexa647 mSA conjugate with VGlut1-SEP [Fig. 1(a)], indicating that mSA-labeled AP-$\mathrm{Nrx}1\beta$ efficiently reaches presynapses. The axonal shaft was almost depleted of staining, probably owing to the fast diffusion of $\mathrm{Nrx}1\beta$ in this compartment.^18,19 Zooming on presynapses to decipher the internal substructure of $\mathrm{Nrx}1\beta$ distribution, we found that $\mathrm{Nrx}1\beta$ exhibits a dual-localization pattern: one population fairly dispersed within the presynaptic membrane and a second population condensed into 1 to 2 nanocluster(s) (mean $1.4\pm 0.18$, 24 synapses) with higher protein density [Figs. 1(b), 1(c), 1(d)]. Using the SR-Tesseler segmentation technique,²⁴ we determined the size of those nanoclusters, which lays in the range of 50 to 200 nm (median $118\mathrm{nm}\pm \mathrm{IQR}$ 80 to $-154\mathrm{nm}$, 37 synapses) [Fig. 1(e)]. These nanoclusters match the size of confinement domains previously observed by uPAINT performed on green fluorescent protein (GFP)-$\mathrm{Nrx}1\beta$ labeled with Atto647N-conjugated anti-GFP nanobody,¹⁸ which most likely correspond to $\mathrm{Nrx}1\beta$ being trapped at presynapses.

Fig. 1

Nanoscale organization of $\mathrm{Nrx}1\beta$ at the presynapse. (a) DIV 15 neurons expressing the presynaptic marker VGlut1-SEP, AP-$\mathrm{Nrx}1\beta$, and BirA^ER were labeled with mSA-Alexa647 and fixed for dSTORM. (b) dSTORM image of an axonal segment with four presynapses and corresponding Voronoï diagram showing $\mathrm{Nrx}1\beta$ enrichment at presynapses, as well as subcluster formation within these enriched regions. (c) Zoom on an $\mathrm{Nrx}1\beta$ subcluster in the polygon representation. (d) Number of $\mathrm{Nrx}1\beta$ subclusters per presynapse. (e) Histogram showing the sizes of $\mathrm{Nrx}1\beta$ subclusters.

2.2.

Difference of Organization Between Nrx1$\beta$, Nlg1, and LRRTM2 at Synapses

To gain insight on the nanoscale organization of postsynaptic adhesion proteins, we imaged Nlg1 and LRRTM2 by dSTORM in DIV 15 neurons (Fig. 2). Nlg1 was both present in the shaft and mildly enriched in dendritic spines, where it exhibited a fairly homogeneous distribution [Fig. 2(a)]. In contrast, LRRTM2 was almost depleted from the shaft and highly enriched in dendritic spines, where it formed one main central cluster with higher protein density [Fig. 2(b)]. To compare the nanoscale organization of Nlg1 and LRRTM2, we used SR-Tesseler²⁴ to define a local molecular density parameter $d$ expressed in a log scale. For Nlg1, the distribution of molecule density exhibited a single population [Fig. 2(d)], reflecting the fact that Nlg1 covers a continuum of organizational states, from a very diffuse state in the shaft to the formation of small synaptic and extrasynaptic aggregates.¹⁸ In contrast, the $d$ distribution for LRRTM2 was broader and composed of two populations [Fig. 2(e)]: a first population of lower density revealing a relative depletion of LRRTM2 from the shaft compared to synapses and a second population of much higher protein density, likely corresponding to the highly enriched synaptic LRRTM2 domains.¹⁸ Interestingly, when applying the same method to presynapses, $\mathrm{Nrx}1\beta$ also showed a bimodal $d$ distribution [Fig. 2(f)], with a first large peak of lower density likely representing $\mathrm{Nrx}1\beta$ present in the axon shaft and at presynapses, and a second peak much shifted to the right, matching the enriched protein density in nanoclusters [Fig. 2(c)]. Altogether, these results suggest that $\mathrm{Nrx}1\beta$ displays two modes of organization: a diffuse state resembling Nlg1 organization and a concentrated state reminiscent of LRRMT2 or even more enriched.

Fig. 2

Differential organization of $\mathrm{Nrx}1\beta$, Nlg1, and LRRTM2 in synaptic compartments. (a, c, and e) Color-coded images showing the normalized molecular densities in log scale for (a) Nlg1, (b) LRRTM2, and (c) $\mathrm{Nrx}1\beta$ obtained from dSTORM images of AP-tagged proteins labeled with mSA-Alexa647. Note the relatively homogenous localization of Nlg1 (green and yellow values) throughout the shaft and dendritic spines, as compared to $\mathrm{Nrx}1\beta$ and LRRTM2 which are highly segregated in pre- and postsynaptic compartments, respectively (blue in the shaft and red in synapses). (d, e, and f) Corresponding distributions of the relative molecular density in semilog plot for Nlg1, LRRTM2, and $\mathrm{Nrx}1\beta$. Statistics: Nlg1 (3 cells, 3,653,239 localizations, $R^2=\mathrm{0,998}$), LRRTM2 (5 cells, 2,971,150 localizations, $R^2=\mathrm{0,992}$), and $\mathrm{Nrx}1\beta$ (5 cells, 1,582,629 localizations, $R^2=\mathrm{0,988}$). Note the single population for Nlg1, and the dual populations for LRRTM2 and $\mathrm{Nrx}1\beta$.

2.3.

Nrx1$\beta$ Organizational Patterns Display Close Similarities to Both Nlg1 and LRRTM2

To compare the nanoscale organization of presynaptic $\mathrm{Nrx}1\beta$ facing that of postsynaptic Nlg1 and LRRTM2, we defined the enrichment factor $R$, which measures the normalized molecular concentration on a linear scale. This enrichment factor was then used as a threshold to highlight the enriched areas with respect to nonenriched ones [Fig. 3(a)].

Fig. 3

The nanoscale organization of $\mathrm{Nrx}1\beta$ lies between that of Nlg1 and LRRTM2. (a) Color-coded images showing the enrichment factor $R$ in linear scale for Nlg1, LRRTM2 in two dendritic spines, and $\mathrm{Nrx}1\beta$ in a presynaptic terminal. Note the mild synaptic enrichment of Nlg1 compared to LRRTM2, which is highly concentrated in a single large domain. $\mathrm{Nrx}1\beta$ shows both a diffuse distribution throughout the presynapse, together with small and highly enriched nanoclusters. (b) Ratio of the enriched area relative to the whole neuronal area considered $A(R)$, as a function of the enrichment factor $R$, for the three proteins. $A(R)$ drops quickly as the enrichment factor is increased, and the curve for $\mathrm{Nrx}1\beta$ lies between those representing Nlg1 and LRTTM2. (c) Ratio of the number of single-molecule localizations in enriched areas $N(R)$, relative to the total number of localizations in the neuronal area, as a function of the enrichment factor $R$, for the three proteins. In this quantification, the curve for $\mathrm{Nrx}1\beta$ is closer to that of LRTTM2.

We then graphed two parameters as a function of $R$: the fraction of neuronal area occupied by enriched regions $A(R)$ [Fig. 3(b)] and the fraction of the number of localizations inside those enriched regions $N(R)$ [Fig. 3(c)]. Interestingly, $A(R)$ was significantly lower for LRRTM2 than for Nlg1, confirming that LRRTM2 is segregated in smaller regions in the spines than Nlg1. When $A(R)$ becomes equivalent for Nlg1 and LRRTM2 for higher $R$ [Fig. 3(b)], $N(R)$ remains two- to threefold higher for LRRTM2 compared to Nlg1 [Fig. 3(c)], indicating that these LRRTM2 segregated regions are much denser.

On the other hand, the curve $A(R)$ for $\mathrm{Nrx}1\beta$ is situated between those of Nlg1 and LRRTM2. $A(R)$ falls deeper than the curve corresponding to Nlg1 due to the fact that the diffuse $\mathrm{Nrx}1\beta$ population covers a relatively small presynaptic surface area compared to Nlg1. In parallel, the curve $A(R)$ for $\mathrm{Nrx}1\beta$ remains higher than that of LRRTM2, because LRRTM2 is segregated in small regions in the spines and lacks a diffuse population. Finally, while $\mathrm{Nrx}1\beta$ is slightly more concentrated than LRRTM2 for low $R$, $N(R)$ becomes equivalent for large $R$. This suggests that $\mathrm{Nrx}1\beta$ has a dual organization, one part being fairly diffuse and disappearing when reaching intermediate $R$ and the other more concentrated with a magnitude equivalent to LRRTM2.

4.1.

Deoxyribose Nucleic Acid Plasmids

The AP-Nlg1, AP-$\mathrm{Nrx}1\beta$, and ${\mathrm{BirA}}^{\mathrm{ER}}$ constructs^17,27 were kind gifts from A. Ting (MIT, Boston). AP-LRRTM2 was generated using the In-Fusion HD Cloning kit (Clontech), replacing the myc-tag from myc-LRRTM2²⁶ (J. de Wit, Leuwen, Belgium) by the AP-Tag (amino acid sequence GLNDIFEAQKIEWHE) as described.¹⁸ SEP-VGlut1 was a kind gift from D. Perrais (Interdisciplinary Institute for Neuroscience, Bordeaux). mSA was subcloned from the previously described pRSET-A vector generously given by S. Park (Buffalo University, New York)^36,37 into pET-IG, a homemade vector derived from pET-24 (Novagen) and engineered to incorporate after the start codon a decahistidine tag immediately followed by a Tobacco Etch Virus cleavage site (-ENLYFQS-) and no tag on the $C$-terminus.

4.2.

Streptavidin Monomers Production and Coupling to Fluorophores

mSA was produced as previously described.¹⁸ Briefly, mSA, encoded in the pET-IG-mSA vector, was expressed by autoinduction in Escherichia coli BL21 codon plus™ (DE3)-RIL for 12 h at 16°C. Following lysis of the bacteria in denaturing conditions, the protein was purified by immobilized metal ion affinity chromatography with HIS-Buster Cobalt Affinity gel (AMOCOL) and refolded in presence of reduced and oxidized glutathione. The refolded protein was concentrated and coupled to Alexa Fluor 647 NHS ester following the recommended procedures from the manufacturer. Excess dye was removed using Sephadex G-25 medium, the conjugate was further purified to homogeneity by size exclusion chromatography, and the labeled protein was aliquoted and stored at $-80°\mathrm{C}$ until use.

4.3.

Cell Culture and Electroporation

Pregnant female rats were purchased weekly (Janvier Labs, Saint-Berthevin, France). Animals were handled and euthanized according to European ethical rules. Dissociated hippocampal neurons from E18 Sprague-Dawley rats embryos were prepared as described³⁸ and electroporated with the Amaxa system (Lonza), using 500,000 cells per cuvette. The following plasmid combinations were used: (GFP or SEP-VGlut1) + ${\mathrm{BirA}}^{\mathrm{ER}}$ + (AP-Nlg1, AP-LRRTM2, or AP-$\mathrm{Nrx}1\beta$) (1.5:1.5:1.5) $\mu \mathrm{g}$ DNA. Electroporated neurons were resuspended in minimal essential medium supplemented with 10% horse serum (MEM-HS) and plated on 18-mm coverslips previously coated with $1\mathrm{mg}{\mathrm{ml}}^{-1}$ polylysine for 2 h, at a concentration of 50,000 cells per coverslip. Three hours after plating, coverslips were flipped onto 60-mm dishes containing a glial cell layer in neurobasal medium supplemented with 2 mM L-glutamine and $1\times$ NeuroCult SM1 Neuronal supplement (STEMCELL technologies), and cultured for 2 weeks at 37°C and 5% ${\mathrm{CO}}_2$. Astrocytes feeder layers were prepared from the same embryos, plated between 20,000 and 40,000 cells per 60-mm dish and cultured in MEM (Fisher Scientific, Cat. No. 21090-022) containing $4.5{\mathrm{gl}}^{-1}$ glucose, 2-mM L-glutamine, and 10% horse serum (Invitrogen) for 14 days.

4.4.

Direct Stochastic Optical Reconstruction Microscopy

Primary cultured neurons expressing AP-Nlg1, AP-LRRTM2, or AP-Nrx1b were surface-labeled with a high concentration (100 nM) of mSA-Alexa647 in Tyrode solution (15 mM D-glucose, 108 mM NaCl, 5 mM KCl, 2 mM ${\mathrm{MgCl}}_2$, 2 mM ${\mathrm{CaCl}}_2$, and 25 mM HEPES, pH 7.4) containing 1% globulin-free BSA (Sigma) for 10 min, rinsed and fixed with 4% PFA-0.2% glutaraldehyde in PBS for 10 min at room temperature. Before dSTORM acquisitions, the samples were incubated in a solution of PBS containing 1:1000 fluorescent beads (Tetraspec) used as markers for image registration. dSTORM imaging of cultured neurons was performed using an inverted motorized microscope (Nikon Ti-Eclipse, Japan) equipped with a $100\times /1.49$ NA PL-APO objective and a perfect focus system, allowing long acquisition in oblique illumination mode. Both the ensemble and single-molecule fluorescence were collected by using a quad-band dichroic filter (Di01-R405/488/561/635, Semrock, New York). The fluorescence was collected using a sensitive EMCCD (Evolve, Photometrics, Arizona). Single-molecule localization and reconstruction were performed online with automatic feedback control of the lasers using WaveTracer module, enabling optimal single-molecule density during the acquisition.³⁹ The acquisition and localization sequences were driven by MetaMorph software (Molecular Devices, California) in streaming mode at $50\text{frames}/\mathrm{s}$ (20-ms exposure time) using an area equal to or less than a $256\times 256\text{pixels}$ region of interest. dSTORM superresolution images were reconstructed from 40,000 frames.

4.5.

Image Reconstruction and Analysis

Image stacks were analyzed using a custom plugin running on MetaMorph software based on wavelet segmentation.^39,40 It allows reconstructing the superresolution images by summing the positions of localized single molecules into a single image and exporting files containing the spatial coordinates of each localization. Fluorescent beads (Tetraspec) were used as fiduciary markers for image registration.

4.6.

Protein Quantification Using Tessellation-Based Analysis

We used SR-Tesseler²⁴ to quantify the molecular organization of the three synaptic adhesion proteins. Single-molecule localization coordinates were used to compute a Voronoï tessellation in order to partition the image space in polygons of various sizes centered on each localized molecule. Using this space-partitioning framework, first-rank densities ${\delta }_i^1$ of the molecules were computed²⁴ and density maps were generated by texturing the Voronoï polygons with ${\delta }_i^1$ values. Segmentations of the mSA-labeled AP-$\mathrm{Nrx}1\beta$ presynapses were performed by applying a threshold of twice the average density $\delta$ of the whole axonal region. Then subcluster organization of the $\mathrm{Nrx}1\beta$ distribution was obtained by applying a threshold of twice the average density of each presynapse. All selected neighboring molecules were merged together to segment presynapses and subclusters, and the size parameters were extracted by principal component analysis.

To determine the nanoscale organization of the three proteins, we first segmented the diffracted-limited low-resolution images using intensity-based thresholding in MetaMorph software. The neuronal regions $N$ were identified by segmenting the localizations inside those masks. All subsequent analyses were performed exclusively on the localizations belonging to $N$. For each protein, normalization of the density distribution was achieved by dividing the density ${\delta }_i^1$ of each molecule by the median ${\tilde{\delta }}^1$ of all ${\delta }_i^1$. The enrichment factor $R$ was defined as the ratio between the molecule density ${\delta }_i^1$ and the average density ${\delta }^N$ of the neuronal region. By varying $R$ (from 0 to 20), we determined enriched regions (for $R>0$) with a given area $A(R)$ and a number of localization $N(R)$. $A(R)$ reflects the surface occupation for ${\delta }_i^1\ge R\times {\delta }^N$ and $N(R)$ the number of localizations within this area.