title: A novel synaptic junction preparation for the identification and characterization of cleft proteins.
abstract: Identification of synaptic cleft components has been hampered by the lack of a suitable preparation enriched in synaptic junctions devoid of adjoining peripheral membranes. Prior strategies for the isolation of synaptic junctions, relying on detergents for the removal of peripheral membranes, resulted in substantial loss of membranes lining the cleft. Here, a novel, detergent-free method is described for the preparation of a synaptic junction (SJ) fraction, using phospholipase A2. Limited digestion of synaptic plasma membrane (SPM) fraction with phospholipase A2 followed by centrifugation over a sucrose cushion results in selective removal of membranes peripheral to the cleft while junctional membranes remain relatively intact as observed by electron microscopy. Enrichment in synaptic junctional structures and loss of membranes peripheral to the junctional area are further verified by demonstrating enrichment in PSD-95 and loss in mGluR5, respectively. The SJ fraction is enriched in neuroligins and neurexins, in agreement with immuno-electron microscopy data showing their selective localization to the junctional area. Among additional cell adhesion molecules tested, N-cadherin and specific isoforms of the SynCAM and SALM families also show marked enrichment in the SJ fraction, suggesting preferential localization at the synaptic cleft while others show little enrichment or decrease, suggesting that they are not restricted to or concentrated at the synaptic cleft. Treatment of the SJ fraction with glycosidases results in electrophoretic mobility shifts of all cell adhesion molecules tested, indicating glycosylation at the synaptic cleft. Biochemical and ultrastructural data presented indicate that the novel synaptic junction preparation can be used as a predictive tool for the identification and characterization of the components of the synaptic cleft.
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Amelia
authors.identifier: http://orcid.org/0000-0001-8533-4293
authors.initials: A
authors.lastname: Burch
authors.affiliation: EM Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Jung-Hwa
authors.initials: JH
authors.lastname: Tao-Cheng
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Ayse
authors.initials: A
authors.lastname: Dosemeci
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D054467:Phospholipases A2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D064529:Receptor, Metabotropic Glutamate 5 / Q000378:metabolism; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D064529:Receptor, Metabotropic Glutamate 5; D054467:Phospholipases A2