Synapses are specialized adhesion sites between neurons that are connected by

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members. dimers (Troyanovsky et al 2007 Harrison et al 2010 or from Oxcarbazepine monomers (Sivasankar et al 2009 Zhang et al 2009 Similarly the contributions of assembly of Ig proteins to their adhesive interactions remain unclear. This contrasts with the detailed structural views of Ig dimers that can assemble in a zipper-like manner to form extended structures as shown for NCAM and TAG-1 (Freigang et al 2000 Soroka et al 2003 or assemble into separately spaced dimers through horseshoe-shaped Ig arrangements such as L1 and Dscam (Meijers et al 2007 He et al 2009 To gain molecular insight into the interactions that organize the synaptic cleft HNPCC we have analysed the assembly steps of Oxcarbazepine SynCAM 1 the founding member of a family of four Ig superfamily proteins that are most prominently expressed in brain (Biederer 2006 Thomas et al 2008 SynCAM proteins contain three extracellular Ig-like domains one transmembrane region and a short cytoplasmic tail with protein interaction motifs predicted to bind cytoskeletal regulators and scaffolding molecules. SynCAM 1 is already expressed in neurons prior to synapse formation and its rapid adhesive assembly at axo-dendritic contacts precedes synapse development (Stagi et al 2010 Subsequently SynCAM 1 engages in homo- and heterophilic adhesive interactions Oxcarbazepine that induce neurons in the central nervous system to assemble pre- but not Oxcarbazepine post-synaptic specializations (Biederer et al 2002 Nam and Chen 2005 Fogel et al 2007 Robbins et al 2010 In the peripheral nervous system SynCAM proteins also mediate interactions of axons with myelinating Schwann cells (Maurel et al 2007 Spiegel et al 2007 Analysing SynCAM adhesion complexes between neurons we now show that SynCAM 1 is clustered in dendrites of developing neurons prior to synaptic contact. We find that the extracellular Ig-like domains 2 and 3 of SynCAM 1 can self-assemble independently of adhesive extracellular interactions or intracellular scaffolds. Notably these interactions of SynCAM 1 promote its adhesive binding in heterologous cells and at axo-dendritic contacts between immature neurons. In addition lateral SynCAM 1 interactions increase its ability to recruit cognate SynCAM binding partners across the nascent synaptic cleft and to induce pre-synaptic specializations. At mature post-synaptic sites the oligomerization of SynCAM 1 then contributes to restricting the size of synaptic specializations. These results identify the lateral self-assembly of the Ig protein SynCAM 1 as a novel determinant in the (d.i.v.) using antibodies against an extracellular epitope. Neuronal cell bodies and dendrites were labelled using antibodies against MAP2 (microtubule-associated protein 2). We found that endogenous SynCAM 1 protein appeared in clusters on MAP2-positive dendrites (Figure 1A left). SynCAM 1 signal was absent in hippocampal cultures from SynCAM 1 knockout mice (Figure 1A right). We next addressed the mobility of these SynCAM 1 clusters by imaging a SynCAM 1 construct carrying an extracellular insertion of the pH-sensitive GFP variant pHluorin. This construct allows to selectively visualize the surface pool of this membrane protein (Stagi et al 2010 SynCAM 1-pHluorin was clustered on the neurites Oxcarbazepine similar to the endogenous protein and these packets were mostly immobile (Figure 1B). Figure 1 SynCAM 1 assembles into discrete clusters in the absence of adhesive contact. (A) SynCAM 1 forms dendritic clusters prior to the onset of synaptogenesis. Dissociated hippocampal wild-type mouse neurons (left) were labelled live at 5 d.i.v. with antibodies … To gain biochemical insight into SynCAM 1 clustering we treated SynCAM 1-expressing COS7 cells with the membrane impermeable 11 ? long cross-linker bis-sulfosuccinimidyl suberate (BS3). Cells were physically separated.