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Fluorescence bioimaging techniques to analyze signal transduction dynamics in living cells

Intracellular signal transduction consists of protein-protein interaction, posttranslational modification including phosphorylation, protein conformational conversions and subsequent change in enzymatic activity, which are precisely controlled in living cells. Moreover, spatiotemporal precision in the regulation is required for proper expression of cellular phenotypes, including protein expression, gene expression, morphological changes, and motility. Fluorescence bioimaging contributes to both qualitative and quantitative analyses of such phenomena.
In our laboratory, the following fluorescence bioimaging techniques are recruited in order to visualize the dynamics of the above cellular events.


Fluorescent protein

Green fluorescent protein (GFP) is a fluorescent protein that was isolated from the luminous organ of the jellyfish Awquorea victoria by Dr. Osamu Shimomura. Nowadays, GFP is widely applied for visualization of protein dynamics or cellular environmental change, due to its convenience of transduction into cells; fluorescent protein cab be introduced into cells by transfection since its cDNA was isolated in 1992.



Förster resonance energy transfer (FRET) is a phenomenon of radiationless excitation energy transfer between fluorescent molecules.
In GFP-based FRET assay, CFP (cyan-emitting mutant) and YFP (yellow fluorescence) are widely used. When CFP and YFP are close to each other, FRET from CFP (donor) to YFP (acceptor) occurs. This principle can be applied to the detection of protein-protein interaction or protein conformational change with high sensitivity and quantitativity in living cells.





GFP is 240 amino acids long and folds into a cylindrical caged structure of β- barrel, which is essential for its function, fluorescence emission. As other functional proteins, GFP can be divided into two fragments that individually possess no fluorescence but that can be reconstituted as a functionally active complex when they are brought in proximity to each other. Fusing the fragments to partners that interact with each other enhances the efficacy of their assembly (Figure 2). Therefore, the fluorescence intensity emitted by the complex can be used to monitor bimolecular interactions. This method is named as “bimolecular fluorescence complementation” (BiFC).
An advantage of BiFC is that the protein-protein interaction is indicated by an increase in fluorescence intensity of single color. Therefore, in the case of GFP-based BiFC you can monitor the protein-protein interaction by using a FITC filter set (the most distributed filter set) equipped conventional fluorescence microscopy. In addition, multiple protein interactions can be simultaneously observed in a single cell using a multicolor BiFC analysis.




【Related paper】

  • Visualization of Ras-PI3K interaction in the endosome using BiFC. K. Tsutsumi, Y. Fujioka, M. Tsuda,H. Kawaguchi, Y. Ohba. Cell Signal. 21(11): 1672-1679 (2009)