Bead aggregation assay to demonstrate the clustering of postsynaptic proteins by the NGL family of cell adhesion molecules
Related Journal & Article Information
Journal: Nature Neuroscience
Article Title: NGL family PSD-95–interacting adhesion molecules regulate excitatory synapse formation
Introduction
Bead-induced direct aggregation of neuroligin on the surface membrane of neuronal dendrites induced the clustering of neuroligin and various postsynaptic proteins [1]. We employed this assay to demonstrate that direct aggregation of NGL, a family of cell adhesion molecules that associates with the netrin-G family of cell adhesion molecules and the postsynaptic scaffolding protein PSD-95, on the dendritic surface induces coclustering of excitatory postsynaptic proteins including PSD-95, GKAP, Shank, and NMDA glutamate receptors.
Materials
Reagents
Equipment
Procedure
Neuron transfection
1. Transfect cultured neurons with a form of NGL-2 in which EGFP was tagged to the N terminus (EGFP-NGL-2) at DIV 14, and incubate for 2 days.
Preparation of EGFP antibody-coated beads
2. Mix 5 μg of biotin-conjugated EGFP antibodies (Rockland) and 2 μL of neutravidin-conjugated FluoSphere beads (Molecular Probes; 1 μm diameter), and incubate for 1 h.
3. Increase the volume of the mixture to 1 mL by adding Hank’s balanced salt solution (HBSS), and centrifuge at 13,000 rpm for 1 min in a microcentrifuge.
4. Carefully remove the supernatant, and resuspend the precipitates in 100 μL of conditioned media where neurons were growing.
Bead-induced aggregation of NGL-2
5. Place the coverslips containing transfected neurons (DIV16) face-up on 6-well (or larger) dishes. Save the conditioned media for Step 7.
6. Add 100 μL of the resuspended beads onto the neurons and incubate at 37 oC for 30 min.
7. Wash the neurons twice with HBSS, and place the coverslip back into the culture media.
8. After 24 h of incubation, fix and immunostain the neurons with primary antibodies against various synaptic proteins and fluorophore-conjugated secondary antibodies.
Image acquisition and quantitation
9. Capture Z-stacked images by confocal immunofluorescence microscopy as well as DIC imaging.
10. For quantitation, manually trace the boundaries of the beads. Copy a boundary to a nearby dendritic area for normalization.
11. An average immunofluorescence intensity of a synaptic marker from a bead area was measured and normalized to that from a nearby control area.
Troubleshooting
Critical Steps
Anticipated Results
References
1. Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W. & Craig, A. M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119, 1013-26 (2004).
Acknowledgements
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