Bead aggregation assay to demonstrate the clustering of postsynaptic proteins by the NGL family of cell adhesion molecules

doi:10.1038/nprot.2006.293

Method Article

Bead aggregation assay to demonstrate the clustering of postsynaptic proteins by the NGL family of cell adhesion molecules

https://doi.org/10.1038/nprot.2006.293

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Biochemistry

Computational biology and bioinformatics

Neuroscience

Biological techniques

Cell biology

Developmental biology

neuroligin

neuronal dendrites

NGL

cell adhesion

PSD-95

postsynaptic proteins

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.

Neuron transfection

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

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.
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.
Carefully remove the supernatant, and resuspend the precipitates in 100 μL of conditioned media where neurons were growing.

Bead-induced aggregation of NGL-2

Place the coverslips containing transfected neurons (DIV16) face-up on 6-well (or larger) dishes. Save the conditioned media for Step 7.
Add 100 μL of the resuspended beads onto the neurons and incubate at 37 ^oC for 30 min.
Wash the neurons twice with HBSS, and place the coverslip back into the culture media.
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

Capture Z-stacked images by confocal immunofluorescence microscopy as well as DIC imaging.
For quantitation, manually trace the boundaries of the beads. Copy a boundary to a nearby dendritic area for normalization.
An average immunofluorescence intensity of a synaptic marker from a bead area was measured and normalized to that from a nearby control area.

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).

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Bead aggregation assay to demonstrate the clustering of postsynaptic proteins by the NGL family of cell adhesion molecules

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Bead aggregation assay to demonstrate the clustering of postsynaptic proteins by the NGL family of cell adhesion molecules

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