Biochemical and Biophysical Research Communications
Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity
Section snippets
Materials and methods
Plasmid construction. The SynCAM ECD hFc construct was generated by inserting the extracellular domain of SynCAM in pREP10 vector containing the Fc domain of human IgG (a gift from RM Mege, INSERM, Paris). The extracellular domain of SynCAM (aa 1–377) was amplified from a pCMV-SynCAM1 (a gift from T. Biederer, UT Southwestern) with primers sense 5′-AGATCTGCAGGTGCCCGACATGGCGAGT-3′ and anti-sense 5′-AGATCTACTTACCTGCGTGGTCCACTGCCCCAAT-3′, each containining a BglII site. This 1160 pb PCR product was
Production and characterization of a SynCAM-Fc chimera
Because SynCAM extracellular domains seem to form quite robust homophilic interactions, both in affinity chromatography [4] and cell aggregation assays [3], we decided to label SynCAM receptors in neurons using SynCAM itself as a ligand. We first produced a plasmid containing the SynCAM extracellular sequence fused in C-terminal with the constant fragment of human immunoglobulin G (Fig. 1A). We expressed this construct in HEK cells, harvested the conditioned medium, and purified the secreted
Acknowledgments
We thank M. Lambert and R.M. Mège for the purification protocol, T. Biederer for the gift of the SynCAM plasmid, D. Bouchet and B. Tessier for help with cell culture, Grégory Giannone, M. Heine and C. Bats for advice on Quantum Dots coupling protocol, L. Cognet and M. Renner for the Mathlab program.
References (14)
Bioinformatic characterization of the SynCAM family of immunoglobulin-like domain-containing adhesion molecules
Genomics
(2006)- et al.
The tumor suppressor protein TSLC1 is involved in cell–cell adhesion
J. Biol. Chem.
(2002) - et al.
Distribution of RA175/TSLC1/SynCAM, a member of the immunoglobulin superfamily, in the developing nervous system
Brain Res. Dev. Brain Res.
(2005) - et al.
Weak effect of membrane diffusion on the rate of receptor accumulation at adhesive contacts
Biophys. J.
(2005) - et al.
Nectin-like molecule-1/TSLL1/SynCAM3: a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes
J. Cell Sci.
(2005) - et al.
SynCAM, a synaptic adhesion molecule that drives synapse assembly
Science
(2002) - et al.
Selective capability of SynCAM and neuroligin for functional synapse assembly
J. Neurosci.
(2005)
Cited by (12)
Molecular composition of developing glutamatergic synapses
2020, Synapse Development and Maturation: Comprehensive Developmental NeuroscienceDynamics, nanoscale organization, and function of synaptic adhesion molecules
2018, Molecular and Cellular NeuroscienceCitation Excerpt :Synaptic Cell Adhesion Molecules (SynCAMs) are a group of adhesion proteins that belong to the immunoglobulin super-family, known to modulate synapse formation. SynCAM1 was originally identified as a synaptogenic protein in a co-culture assay (Biederer et al., 2002), and later shown using biomimetic substrates to exhibit a preference for inducing pre-synapse formation (Breillat et al., 2007; Czöndör et al., 2013). SynCAMs exist as four isoforms (SynCAM1,2,3,4), which display both homophilic and heterophilic trans-binding activity (Fogel et al., 2007), and can form cis-homodimers.
Molecular composition of developing glutamatergic synapses
2013, Cellular Migration and Formation of Neuronal Connections: Comprehensive Developmental NeuroscienceMolecular Composition of Developing Glutamatergic Synapses
2013, Cellular Migration and Formation of Neuronal ConnectionsNeurexin/neuroligin interaction kinetics characterized by counting single cell-surface attached quantum dots
2009, Biophysical JournalCitation Excerpt :SDS-PAGE and immunoblotting with a mouse antibody against human Fc (Jackson Immunoresearch, West Grove, PA) was performed as described (2). The Fc fusion proteins spontaneously assemble into dimers due to the presence of disulfide bonds in the Fc fragment (18) (Fig. 1b). We also produced monomeric Nrx1β-Fc by treating the purified protein preparation with 10 mM dithiothreitol (DTT) for 30 min at room temperature, then centrifuging at 14,000 rpm for 2 h in Centricon filtering units (Millipore, Billerica, MA).
Redundant postsynaptic functions of SynCAMs 1–3 during synapse formation
2017, Frontiers in Molecular Neuroscience