Role of regulatory C-terminal motifs in synaptic confinement of LRRTM2.

Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain.

Dynamics, nanoscale organization, and function of synaptic adhesion molecules.

Synaptic adhesion molecules not only provide a physical link between pre- and post-synaptic membranes, but also contribute to synaptic differentiation and plasticity by organizing functional elements, in particular neurotransmitter receptors. The wealth of existing adhesive protein families including many isoforms and splice variants, calls for systematic identification of the levels and exchange rates of each of those protein members at specific synapse types. Complementary to electron microscopy to identify individual synaptic contacts and biochemistry to reveal protein-protein interactions, recent super-resolution light microscopy methods combined with appropriate fluorescent labeling provide a way to measure the dynamics and sub-micron organization of selective molecular components, and their inter-relations at the synapse. In this review, we summarize current knowledge on the dynamics, nanoscale localization, and function of key synaptic adhesion complexes.

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.

Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons.

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior.

MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

Biophysical mechanisms underlying the membrane trafficking of synaptic adhesion molecules.

Adhesion proteins play crucial roles at synapses, not only by providing a physical trans-synaptic linkage between axonal and dendritic membranes, but also by connecting to functional elements including the pre-synaptic neurotransmitter release machinery and post-synaptic receptors. To mediate these functions, adhesion proteins must be organized on the neuronal surface in a precise and controlled manner. Recent studies have started to describe the mobility, nanoscale organization, and turnover rate of key synaptic adhesion molecules including cadherins, neurexins, neuroligins, SynCAMs, and LRRTMs, and show that some of these proteins are highly mobile in the plasma membrane while others are confined at sub-synaptic compartments, providing evidence for different regulatory pathways. In this review article, we provide a biophysical view of the diffusional trapping of adhesion molecules at synapses, involving both extracellular and intracellular protein interactions. We review the methodology underlying these measurements, including biomimetic systems with purified adhesion proteins, means to perturb protein expression or function, single molecule imaging in cultured neurons, and analytical models to interpret the data. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins.

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell-cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.

TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering.

Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.

A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation.

To better understand the molecular mechanisms by which early neuronal connections mature into synapses, we examined the impact of neuroligin-1 (Nlg1) phosphorylation on synapse differentiation, focusing on a unique intracellular tyrosine (Y782), which differentially regulates Nlg1 binding to PSD-95 and gephyrin. By expressing Nlg1 point mutants (Y782A/F) in hippocampal neurons, we show using imaging and electrophysiology that Y782 modulates the recruitment of functional AMPA receptors (AMPARs). Nlg1-Y782F impaired both dendritic spine formation and AMPAR diffusional trapping, but not NMDA receptor recruitment, revealing the assembly of silent synapses. Furthermore, replacing endogenous Nlg1 with either Nlg1-Y782A or -Y782F in CA1 hippocampal neurons impaired long-term potentiation (LTP), demonstrating a critical role of AMPAR synaptic retention. Screening of tyrosine kinases combined with pharmacological inhibitors point to Trk family members as major regulators of endogenous Nlg1 phosphorylation and synaptogenic function. Thus, Nlg1 tyrosine phosphorylation signaling is a critical event in excitatory synapse differentiation and LTP.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

Molecular determinants for the strictly compartmentalized expression of kainate receptors in CA3 pyramidal cells.

Distinct subtypes of ionotropic glutamate receptors can segregate to specific synaptic inputs in a given neuron. Using functional mapping by focal glutamate uncaging in CA3 pyramidal cells (PCs), we observe that kainate receptors (KARs) are strictly confined to the postsynaptic elements of mossy fibre (mf) synapses and excluded from other glutamatergic inputs and from extrasynaptic compartments. By molecular replacement in organotypic slices from GluK2 knockout mice, we show that the faithful rescue of KAR segregation at mf-CA3 synapses critically depends on the amount of GluK2a cDNA transfected and on a sequence in the GluK2a C-terminal domain responsible for interaction with N-cadherin. Targeted deletion of N-cadherin in CA3 PCs greatly reduces KAR content in thorny excrescences and KAR-EPSCs at mf-CA3 synapses. Hence, multiple mechanisms combine to confine KARs at mf-CA3 synapses, including a stringent control of the amount of GluK2 subunit in CA3 PCs and the recruitment/stabilization of KARs by N-cadherins.

Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy.

The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-[Formula: see text] ([Formula: see text]), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe ([Formula: see text]) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and [Formula: see text] in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of [Formula: see text] interacts transsynaptically with Nlg1 and the other part with LRRTM2.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin.

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1ß, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.

The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors.

The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.

Role of the neuronal K-Cl co-transporter KCC2 in inhibitory and excitatory neurotransmission.

The K-Cl co-transporter KCC2 plays multiple roles in the physiology of central neurons and alterations of its function and/or expression are associated with several neurological conditions. By regulating intraneuronal chloride homeostasis, KCC2 strongly influences the efficacy and polarity of the chloride-permeable γ-aminobutyric acid (GABA) type A and glycine receptor (GlyR) mediated synaptic transmission. This appears particularly critical for the development of neuronal circuits as well as for the dynamic control of GABA and glycine signaling in mature networks. The activity of the transporter is also associated with transmembrane water fluxes which compensate solute fluxes associated with synaptic activity. Finally, KCC2 interaction with the actin cytoskeleton appears critical both for dendritic spine morphogenesis and the maintenance of glutamatergic synapses. In light of the pivotal role of KCC2 in the maturation and function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. These include development and activity-dependent modifications both at the transcriptional and post-translational levels. We emphasize the importance of post-translational mechanisms such as phosphorylation and dephosphorylation, oligomerization, cell surface stability, clustering and membrane diffusion for the rapid and dynamic regulation of KCC2 function.