title: Live-cell imaging of receptors around postsynaptic membranes.
abstract: This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein-coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
authors: Hiromitsu
authors: H
authors: Tanaka
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
authors: Shumpei
authors: S
authors: Fujii
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
authors: Tomoo
authors: T
authors: Hirano
mesh_terms: D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009473:Neuronal Plasticity; D061848:Optical Imaging / Q000379:methods*; D017470:Receptors, Glutamate / Q000378:metabolism*
chemical_list: C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D017470:Receptors, Glutamate