Neuronal synapse interaction reconstituted between live cells and supported lipid bilayers

Abstract

In the nervous system, homophilic and heterophilic adhesion molecules participate in the induction and differentiation of presynaptic transmitter release sites. We focus on the heterophilic interaction between postsynaptic neuroligin-1 (Nlg) and presynaptic β-neurexin (Nrx). Nlg has previously been shown to trigger presynaptic differentiation in a Nrx-expressing axon even when presented on a nonneuronal cell or on beads coated with lipid bilayers. We have now developed a new method to measure single molecule and ensemble distribution of Nrx and Nlg at the contact site between a non-neuronal Nrx-expressing cell and a flat supported glycosylphosphoinositol-neuroligin-1 (GPI-Nlg) lipid bilayer and relate them to adhesion as measured by cell migration and gravity dissociation. We find that within minutes after cell-bilayer contact, Nrx accumulates at the contact site and the contact area is expanded. The strength of cell-bilayer adhesion depends on the morphology of Nrx accumulation, with the focal concentration strengthening adhesion. The results suggest that Nlg-Nrx interaction rapidly establishes a weak, but specific, adhesion between dynamic pre-and postsynaptic processes, which may ultimately require additional molecules for synapse stabilization.Adhesion proteins present on the surface of a neuron are instrumental to the recognition and creation of synaptic connections with appropriate partner cells 1-3 . The extracellular domains of those adhesion proteins bind selectively to cognate adhesion proteins in partner cells, whereas their internal domains bind to cytoplasmic scaffolding proteins. Such a transcellular molecular bridge between signaling systems inside the two cells leads to the organization of the neuronal synapse 1-3 . Molecular interactions can be mediated by homophilic adhesion molecules, such as NCAM 4,5 , N-cadherins 6,7 and syncam 8 , or by heterophilic adhesion molecules, such as integrins, or post-synaptic Nlg interacting with presynaptic Nrx 9-13 . Syncam and Nlg-Nrx are the only known pairs whose interactions trigger presynaptic differentiation 8,12,13 . We have focused our attention on the heterophilic interaction between Nlg and Nrx.Nlg and Nrx bind in a calcium-dependent fashion in a stoichiometric ratio of 1:1 (ref. 10). Nonneuronal cells expressing Nrx aggregate with non-neuronal cells expressing Nlg, recruiting Nlg and Nrx to the cell-cell contact 10,13 . In addition, contact between a non-neuronal Nlgexpressing cell and a Nrx-bearing axon of a primary cultured neuron is sufficient to trigger

E-cadherin junction formation involves an active kinetic nucleation process

“…Overall, E-cadherin is unique among the juxtacrine receptor− ligand systems that so far have been studied in detail in hybrid live cell-supported membrane junctions (7,8,(27)(28)(29)(30). Intermembrane E-cadherin adhesion does not appear to follow simple laws of mass action.…”

Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation

“…There are several potential explanations for this finding: (1) although Nrxs have also been reported in dendrites 36 , those molecules are mostly presynaptic; thus, neuronal adhesion to Nlg1-coated dots is mediated essentially by axons, which may not be sufficient to promote strong somato-dendritic adhesion and cell survival; (2) purified Nlg1 protein may be more fragile than Nrx1b and get degraded throughout several days at 37°C; (3) Nlg1 can induce presynaptic differentiation only when it is freely diffusible in lipid membranes and not when immobilized on a substrate 14,37,38 ; and (4) Nlg1, which is assembled as a constitutive dimer through interactions between extracellular AchE-like domains 14,39 , does not function properly when presented as a Nlg1-6His monomer.…”

Biomimetic Nanopatterns as Enabling Tools for Analysis and Control of Live Cells

“…Peptide Receptor Patterning -Supported Lipid Bilayer [96][97][98][99] -E-beam Lithography [56,96] -Dip-Pen Nanolithography [18,35] Nanoscale Ligand Patterning…”