Invited reviewBiophysical mechanisms underlying the membrane trafficking of synaptic adhesion molecules
Introduction
The synapse is a highly crowded and confined environment where thousands of proteins interact with one another to instruct neuronal communication across the brain. Synaptic Cell Adhesion Molecules (CAMs) are the building blocks of neuronal communication, responsible for transmembrane binding allowing contact initiation between neurons, and for signaling mechanisms leading to synapse specification, maintenance, and plasticity. Although some neuronal CAMs have been largely studied such as the homophilic N-Cadherin or heterophilic neurexin-neuroligin (Nrx-Nlg) complexes, understanding how synaptic CAMs are recruited and stabilized at different synapse types to mediate their specific functions, and how biochemical interactions modulate protein motion and membrane organization remains challenging. On the one hand, the high number of CAMs associated with numerous isoforms and splice variants create a tremendous diversity rendering systematic studies difficult. On the other hand, the lack of tools to specifically target these proteins, such as antibodies or derivatives, makes their study challenging in native contexts. Furthermore, the important redundancy in the functions of the different CAMs render most genetic approaches such as knock-out or knock-down strategies difficult to interpret due to compensatory homeostatic mechanisms, highlighting a need for novel tools to acutely probe and/or perturb the function of adhesion molecules. In this review, we focus on the biophysical properties of the main synaptic CAMs and their molecular partners at synapses, to gain insight on how biochemical interactions provide a mechanism for CAM stabilization, turnover, and nanoscale organization, potentially underlying their synaptic function.
This methodologically-oriented review describes sequentially: (1) in vitro characterization of interactions between adhesion proteins; (2) systems combining biomimetic components and primary neurons for the study of synaptogenesis; (3) perturbation approaches to dissect the roles of specific adhesion proteins in vivo; and (4) fluorescence high resolution imaging experiments in live neurons to assess adhesion molecule dynamics in and out of synapses. Given recent extensive reviews in the field (Reissner et al., 2013, Südhof, 2017), our strongest emphasis is on aspects (2) and (4).
Section snippets
Biochemical and proteomics search for adhesion protein interactors at the synapse
Binding partners of adhesion proteins have been traditionally identified on a case-by case basis, by strategies that consist of affinity isolation of putative binders from cell or tissue extracts (affinity chromatography or co-immunoprecipitation experiments) followed by biochemical analysis. For example, Nlg1 was initially isolated as a binding partner of Nrx1β on affinity columns (Ichtchenko et al., 1995) and later shown to be a post-synaptic component interacting in trans with Nrxs (Irie et
Studying synaptic CAM interactions with biomimetic models
As described above, adhesion protein behavior at synapses relies on multiple factors, including the abundance and affinity of extracellular counter receptors and intracellular scaffolds. These factors being impossible to exactly reproduce in vitro, it can be interesting to maintain on one side a native cellular context, and to simplify the environment on the other side. Biomimetic assays where neurons are placed in contact with artificial surfaces containing specific ligands at controlled
Knock-out and knock-down approaches
The most popular approach to interfere with the function of specific adhesion proteins has been the generation of knock-out and knock-in mice. Full triple αNRX1/2/3 KO show major defects in pre-synaptic release (Missler et al., 2003), linked to an impairment of voltage-gated calcium channels that can bind αNrxs (Zhang et al., 2005), while complete conditional KO of all three Nrx1/2/3 genes induces a wide range of synaptic defects that are dependent on synapse type (Chen et al., 2017).
Imaging synaptic CAM interactions in vivo
Studying protein interactions and turnover in living cells is a requisite for understanding key cellular processes. The classical biochemical techniques for detecting protein interactions in cells and tissue, such as immuno-precipitation or pull-down assays, have allowed the identification of many synaptic protein interactions, with the main advantage being that the whole proteome is “fished” for prey identification and endogenous protein interactions are identified. However, transient or
Conclusion and perspectives
As a conclusion, a particular distribution and nanoscale organization of the different types of adhesion proteins is essential for structuring the synapse and organizing its functional components during synapse establishment, maintenance, and/or plasticity. Working in native cellular environments reveals various important biophysical features that cannot be accessed in vitro, such as the oligomeric state of proteins and actual stoichiometry of the interaction complex, the impact of the membrane
Acknowledgements
We thank all members of the Thoumine team for sharing experiments and ideas that fed the concepts of the review. This work received funding from the Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Agence Nationale pour la Recherche (grants « SynAdh » ANR-13-BSV4-0005-01, « SynSpe » ANR-13-PDOC-0012-01, and « Synthesyn » ANR-17-CE16-0028-01), Commission Franco-Américaine (Fulbright program), Conseil Régional Aquitaine («
References (133)
- et al.
Single-molecule fluorescence microscopy of native macromolecular complexes
Curr. Opin. Struct. Biol.
(2016) - et al.
Structures of neuroligin-1 and the neuroligin-1/neurexin-1β complex reveal specific protein-protein and protein-Ca2+Interactions
Neuron
(2007) - et al.
A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins
Neuron
(2005) - et al.
Increasing numbers of synaptic puncta during late-phase LTP: N-cadherin is synthesized, recruited to synaptic sites, and required for potentiation
Neuron
(2000) - et al.
The structure of neurexin 1α reveals features promoting a role as synaptic organizer
Structure
(2011) - et al.
Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2
Neuron
(2007) - et al.
Characterization of the interaction of a recombinant soluble neuroligin-1 with neurexin-1β
J. Biol. Chem.
(2003) - et al.
The macromolecular architecture of extracellular domain of αNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition
Structure
(2010) - et al.
Altered cortical dynamics and cognitive function upon haploinsufficiency of the autism-linked excitatory synaptic suppressor MDGA2
Neuron
(2016) - et al.
Loss of synapse repressor MDGA1 enhances perisomatic inhibition, confers resistance to network excitation, and impairs cognitive function
Cell Rep.
(2017)
Unbiased discovery of glypican as a receptor for LRRTM4 in regulating excitatory synapse development
Neuron
LRRTM2 interacts with Neurexin1 and regulates excitatory synapse formation
Neuron
Structural analysis of the synaptic protein neuroligin and its β-neurexin complex: determinants for folding and cell adhesion
Neuron
Plug-and-play pairing via defined divalent streptavidins
J. Mol. Biol.
Supramolecular organization of NMDA receptors and the postsynaptic density
Curr. Opin. Neurobiol.
Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1
Cell Rep.
Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins
Cell
Lengthening of the stargazin cytoplasmic tail increases synaptic transmission by promoting interaction to deeper domains of PSD-95
Neuron
A novel multiple PDZ domain-containing molecule interacting with N-Methyl-d-aspartateReceptors and neuronal cell adhesion proteins
J. Biol. Chem.
Neuroligin 1: a splice site-specific ligand for beta-neurexins
Cell
Induction of GABAergic postsynaptic differentiation by alpha-neurexins
J. Biol. Chem.
Structural insights into modulation of neurexin-neuroligin trans-synaptic adhesion by MDGA1/neuroligin-2 complex
Neuron
LRRTM2 functions as a neurexin ligand in promoting excitatory synapse formation
Neuron
Crystal structures of β-neurexin 1 and β-neurexin 2 ectodomains and dynamics of splice insertion sequence 4
Structure
Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificity
J. Biol. Chem.
An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers
Neuron
The complexity of PDZ domain-mediated interactions at glutamatergic synapses: a case study on neuroligin
Neuropharmacology
The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function
Structure
Neurexophilin binding to alpha-neurexins. A single LNS domain functions as an independently folding ligand-binding unit
J. Biol. Chem.
CASK functions as a Mg2+-independent neurexin kinase
Cell
Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP
Neuron
Topographic mapping of the synaptic cleft into adhesive nanodomains
Neuron
The specific α-neurexin interactor calsyntenin-3 promotes excitatory and inhibitory synapse development
Neuron
Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins
Proc. Natl. Acad. Sci. U. S. A
InferenceMAP: mapping of single-molecule dynamics with Bayesian inference
Nat. Methods
Models for the specific adhesion of cells to cells
Science
CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses
Nat. Neurosci.
Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons
Proc. Natl. Acad. Sci. Unit. States Am.
SynCAM, a synaptic adhesion molecule that drives synapse assembly
Science
Imaging of molecular surface dynamics in brain slices using single-particle tracking
Nat. Commun.
Quantitative diffusion measurements using the open-source software PyFRAP
Nat. Commun.
α-Neurexins together with α2δ-1 auxiliary subunits regulate Ca 2+ influx through Ca V 2.1 channels
J. Neurosci.
Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling
Proc. Natl. Acad. Sci. U. S. A
Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin
Nat. Commun.
Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy
Neurophotonics
Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin
Nat. Protoc.
Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines
Mol. Biol. Cell
The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization
J. Cell Biol.
Conditional deletion of all neurexins defines diversity of essential synaptic organizer functions for neurexins
Neuron
Structural basis for synaptic adhesion mediated by neuroligin-neurexin interactions
Nat. Struct. Mol. Biol.
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