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.
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Acknowledgements
We thank S. Kawaguchi, Y. Tagawa and E. Nakajima for comments on the manuscript. We thank S. Okabe (Tokyo University) for the PSD95-EGFP expression vector. This research was supported by grants-in-aid for scientific research, Global Centers of Excellence program A06 of Kyoto University; Grants for Excellent Graduate Schools from the Ministry of Education, Culture, Sports, Science and Technology; and from the Takeda Science Foundation in Japan.
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H.T. and T.H. designed the study and wrote the paper. H.T. and S.F. improved the protocol and performed the experiments.
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Integrated supplementary information
Supplementary Figure 1 NRX-coating check.
(a-c) Observation of coated NRX (green) and NLG-HA (magenta) expressed in HEK cells by immunostaining. (b) When NRX is immunostained without permeabilization of the membrane, NRX signal is not detected under a HEK cell (arrowhead). (c) After permeabilization, NRX under a HEK cell is stained, suggesting that tight coupling of NRX and NLG prevents access of the antibody to NRX under a cell. (b-d) NLG-HA-expressing HEK cells often show fan-shaped lamellipodia-like swelling (b,c, arrows) and / or well-extended filopodia (d, arrows). These morphological changes of HEK cells are signs of strong interaction between NRX on the glass surface and NLG in HEK cells. Scale bars, 100 ÎĽm (a), 20 ÎĽm (c).
Supplementary information
Supplementary Figure 1
NRX-coating check. (PDF 119 kb)
High-frequency imaging of GluA1-SEP.
GluA1-SEP (green) exocytosis in the periphery of PSD95-RFP (magenta)-positive area shown in Figure 8b is recorded. The movie starts at 48.8 s after the start of electrical stimulation, and runs in real time. (MOV 242 kb)
Dual-color imaging of GluA1-SEP and PSD95-RFP.
GluA1-SEP (green) exocytosis in the periphery of PSD95-RFP (magenta)-positive area shown in Figure 8d is recorded. The movie starts at 84.6 s after the start of electrical stimulation, and runs in real time. (MOV 1095 kb)
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Tanaka, H., Fujii, S. & Hirano, T. Live-cell imaging of receptors around postsynaptic membranes. Nat Protoc 9, 76–89 (2014). https://doi.org/10.1038/nprot.2013.171
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DOI: https://doi.org/10.1038/nprot.2013.171
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