Fig 1.
Strategy for the isolation of synaptic junctions.
The synaptic cleft is highlighted in gray. ‘Cleft membranes’ are defined as the membranes within the synaptic junctional area, highlighted in red. Membranes peripheral to the synaptic junction are referred to as ‘peripheral membranes’ and are highlighted in blue. Treatment of the SPM fraction with phospholipase A2 is expected to promote preferential removal of peripheral membranes as compared to the relatively occluded cleft membranes.
Table 1.
List of antibodies.
Fig 2.
Assessment of the optimized synaptic junction preparation by electron microscopy.
Four different synaptic junction fractions were evaluated by electron microscopy. Every recognizable synaptic structure with a postsynaptic density (PSD) was counted and classified into one of the four categories. Ideal synaptic junctions devoid of peripheral membranes and without damage to the cleft membranes were identified as ‘intact’. Synaptic junctions with some undigested peripheral membranes were categorized as ‘with peripheral membranes’. Synapses with some damage to the cleft membranes were identified as ‘partial’, while synapses with total loss of membranes were counted as ‘PSDs’. The bars represent percentages for each type of synaptic structure as a mean of four experiments. The error bars represent the standard error of the mean.
Fig 3.
Verification of synaptic junction fraction.
A) Synaptic plasma membrane (SPM) fractions were incubated in presence (+phlA2) or absence (control) of phosopholipase A2 and centrifuged (See Methods). Comparison of Coomassie Blue stained lanes shows dissociation of proteins as a result of phospholipase A2 digestion. B) Top: Immuno-electron microscopy shows immunogold labeling for PSD-95 localized selectively to the synaptic junction region in cultured rat hippocampal neurons. Bottom: Immunoblots with an antibody for PSD-95 show enrichment of PSD-95 in +phlA2 pellet called ‘synaptic junction’ (SJ) fraction compared to parent homogenate (H) and SPM fractions. Equal amounts of protein were loaded into each lane. The relative enrichment of PSD-95 in the SJ fraction, as compared to that in the parent SPM fraction, was estimated as the ratio of peak areas from densitometric scans. The mean enrichment factor (fold enrichment) from seven immunoblots corresponding to four different SJ preparations was 3.2±0.76. C) Immunoblot with an antibody for mGluR5 shows enrichment of mGluR5 in the SPM fraction compared to the parent homogenate fraction, while there are decreased levels of mGluR5 in the SJ fraction compared to parent SPM fraction. Equal amounts of protein were loaded into each lane.
Fig 4.
All tested cell adhesion molecules are glycosylated in the SJ fraction.
Synaptic junction fractions were incubated with (+) or without (-) a cocktail of glycosidases (Sigma). All proteins tested in the SJ fraction show a shift in mobility after treatment with glycosidases.
Fig 5.
Some cell adhesion molecules are enriched in the SJ fraction, suggesting selective localization at the synaptic cleft.
Western immunoblots comparing synaptic junction (SJ) fractions and parent homogenate (H) and synaptic plasma membrane (SPM) fractions. The lower portion of neurexin immunoblot and the upper portion of SynCAM 1/2/3 immunoblot correspond to higher exposure times to allow optimal visualization of all isoforms. Certain proteins including neuroligins, neurexins, N-cadherin, SynCAM 1 and SALM5 show distinctive enrichment. Equal amounts of protein were loaded into each lane. Experiments were repeated at least twice using different SJ fractions, with similar results (S2 Fig).
Fig 6.
Localization of cell adhesion molecules by immuno-electron microscopy.
Electron micrographs show synaptic regions from perfused mouse brain (top left image only) and cultured rat hippocampal neurons. Neuroligin and neurexin labeling is exclusive to the synaptic region, showing postsynaptic and presynaptic preferences respectively. Whereas, NCAM and SynCAM show a broader distribution within the neuron, with preferential labeling at dendritic and axonal plasma membranes, respectively.