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Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices

Abstract

The plasticity of excitatory synapses has conventionally been studied from a functional perspective. Recent advances in neuronal imaging techniques have made it possible to study another aspect, the plasticity of the synaptic structure. This takes place at the dendritic spines, where most excitatory synapses are located. Actin is the most abundant cytoskeletal component in dendritic spines, and thus the most plausible site of regulation. The mechanism by which actin is regulated has not been characterized because of the lack of a specific method for detection of the polymerization status of actin in such a small subcellular structure. Here we describe an optical approach that allows us to monitor F-actin and G-actin equilibrium in living cells through the use of two-photon microscopy to observe fluorescence resonance energy transfer (FRET) between actin monomers. Our protocol provides the first direct method for looking at the dynamic equilibrium between F-actin and G-actin in intact cells.

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Figure 1: Strategy of using FRET to detect equilibrium between G-actin and F-actin.
Figure 2: Emission spectra of HEK293 cell homogenates that express CFP- and YFP-actin individually or together.
Figure 3: Configuration of a two-photon laser scanning microscope.
Figure 4: FRET image of an NIH3T3 cell transfected with CFP- and YFP-actin.
Figure 5: An example of changes in FRET in response to tetanic stimulation.
Figure 6: An example of an acceptor bleaching experiment.

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Acknowledgements

We thank T. Nagai and A. Miyawaki for discussions and sharing resources, S.M. Kwok for sharing spectral data of CFP-actin and YFP-actin and M. Churchill and T. Emery for editing. Supported by RIKEN, The Ellison Medical Foundation and a National Institutes of Health R01 grant (DA017310-01A1) to Y.H.

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Correspondence to Yasunori Hayashi.

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Okamoto, KI., Hayashi, Y. Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices. Nat Protoc 1, 911–919 (2006). https://doi.org/10.1038/nprot.2006.122

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