Abstract
In this chapter, we introduce the combined use of FRET-based biosensors and synaptic markers as an effective tool for studying intracellular signaling pathways in small synaptic terminals of neuronal cells. The approach is based on the unmixing of excitation/emission spectral fingerprints of a FRET donor and acceptor pair, as well as a lipophilic styryl dye, FM1-43, loaded into presynaptic terminals. The destaining of FM1-43 during evoked release provides a map to guide the sampling of fluorescence for FRET analysis. In the example presented here, we measure the temporal dynamics of cAMP at the presynaptic terminal using an intramolecular CFP/YFP-based FRET sensor. However, this methodology can be applied to investigate the spatial and temporal regulation of a variety of signaling processes, as well as dynamic changes in protein–protein interaction.
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Acknowledgements
The authors would like to thank KunHan Lin for invaluable comments on the protocol and I. Herfort for the technical assistance. This work was funded in part by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain.
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Farsi, Z., Woehler, A. (2017). Imaging Activity-Dependent Signaling Dynamics at the Neuronal Synapse Using FRET-Based Biosensors. In: Poulopoulos, A. (eds) Synapse Development. Methods in Molecular Biology, vol 1538. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6688-2_18
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DOI: https://doi.org/10.1007/978-1-4939-6688-2_18
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