Date Published: March 31, 2017
Publisher: Public Library of Science
Author(s): Amelia Burch, Jung-Hwa Tao-Cheng, Ayse Dosemeci, Mohammed Akaaboune.
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.
The synaptic cleft is a ~20 nm gap between pre- and postsynaptic compartments . Structures that traverse the cleft from the pre- to the postsynaptic membrane are revealed by electron microscopy (EM) , . A recent study, using freeze substitution and EM tomography, identified distinct types of these trans-synaptic structures . The structures bridging the cleft are likely formed by synaptic cell adhesion molecules originating from the pre- and postsynaptic sites, respectively. These molecules have key roles in synaptic adhesion and also act as organizing and signaling elements .
SPM fractions were incubated with phospholipase A2, and the samples were then layered on a sucrose cushion and centrifuged to separate lighter membranes from the denser, junctional material. The quality of the synaptic junctional pellets was evaluated by electron microscopy. Every recognizable synaptic structure encountered was scored according to the presence or absence of peripheral membranes and the intactness of cleft membranes (Fig 2, S1 Fig). The SJ preparation protocol was optimized by altering the concentration of phospholipase A2 and adjusting the duration of the reaction. Under the optimized conditions (See Methods), the majority of synaptic material displayed intact, junctional structures. Less than 20% of synaptic material was classified as PSD-like structures devoid of synaptic junctional membranes (Fig 2). The fraction also contained some mitochondrial and membrane contaminants.
The present study describes a novel, detergent-free method for the preparation of a synaptic junction fraction. Ultrastructural observations indicate that treatment of conventional SPM fractions with phospholipase A2 to remove peripheral membranes yields relatively intact synaptic junctions. This method compares favorably to previously proposed strategies based on the use of low concentrations of mild detergents , , . Selective digestion of peripheral membranes as compared to junctional membranes by phospholipase A2 is most likely due to differences in enzyme penetration. Indeed, junctional membranes lining the synaptic cleft are likely to be occluded by cleft material as well as by protein complexes lining the intracellular faces of membranes at the pre- and post-synaptic compartments. However, it should be noted that the synaptic cleft membranes are only less prone, but not immune, to phospholipase digestion. Thus, optimization of the reaction conditions, especially phospholipase concentration, through monitoring by electron microscopy is critical for the success of the preparation.