No 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

Summary Neuroligins (NLGs) and Neurexins (NRXs) are important adhesion molecules that promote synapse formation. Multiple splice variants of NLG and NRX exist, but their specific functions are unclear. Here we report that a surrogate postsynaptic cell expressing full-length NLG triggers slow presynaptic differentiation in a contacting axon. In contrast, a version of NLG-1, which lacks insert B (NLG-1-ΔB), induces rapid presynaptic differentiation, reaching the rate seen at native neuronal synapses. We show that this acceleration is due to removal of the N-linked glycosylation site within insert B. NLG-1ΔB also increases synaptic density at neuro-neuronal synapses more than does full-length NLG-1. Other postsynaptic adhesion proteins, such as N-cadherin, EphB2 and SynCAM-1, alone or in combination with full-length NLG-1 do not trigger fast differentiation, suggesting that rapid presynaptic differentiation depends on a unique interaction of NLG-1ΔB with axonal proteins. Indeed, we find that NLG-1ΔB recruits more axonal α-NRX. Our results suggest that the engagement of α-NRX is a key to rapid induction of synapses at new sites of axo-dendritic contact.

Genetic sequences ported into non-native hosts for synthetic biology applications can gain unexpected properties. In this study, we explored sequences functioning as ribosome binding sites (RBSs) within protein coding DNA sequences (CDSs) that cause internal translation, resulting in truncated proteins. Genome-wide prediction of bacterial RBSs, based on biophysical calculations employed by the RBS calculator, suggests a selection against internal RBSs within CDSs in Escherichia coli, but not those in Saccharomyces cerevisiae. Based on these calculations, silent mutations aimed at removing internal RBSs can effectively reduce truncation products from internal translation. However, a solution for complete elimination of internal translation initiation is not always feasible due to constraints of available coding sequences. Fluorescence assays and Western blot analysis showed that in genes with internal RBSs, increasing the strength of the intended upstream RBS had little influence on the internal translation strength. Another strategy to minimize truncated products from an internal RBS is to increase the relative strength of the upstream RBS with a concomitant reduction in promoter strength to achieve the same protein expression level. Unfortunately, lower transcription levels result in increased noise at the single cell level due to stochasticity in gene expression. At the low expression regimes desired for many synthetic biology applications, this problem becomes particularly pronounced. We found that balancing promoter strengths and upstream RBS strengths to intermediate levels can achieve the target protein concentration while avoiding both excessive noise and truncated protein.