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Biology Articles » Methods & Techniques » Optical Bioimaging: From Living Tissue to a Single Molecule: Single-Molecule Visualization of Cell Signaling Processes of Epidermal Growth Factor Receptor

Abstract
- Optical Bioimaging: From Living Tissue to a Single Molecule: Single-Molecule Visualization of Cell Signaling Processes of Epidermal Growth Factor Receptor

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Optical Bioimaging: From Living Tissue to a Single Molecule: Single-Molecule Visualization of Cell Signaling Processes of Epidermal Growth Factor Receptor

Yasushi Sako1,2,* Junya Ichinose2, Miki Morimatsu2, Kosuke Ohta3, and Takeshi Uyemura3

1Nanobiology Laboratories, Graduate School of Frontier Biosciences, Osaka University,
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2Time’s Arrow and Biosignaling, PRESTO, JST
3Department of Systems and Human Science, Graduate School of Engineering Science, Osaka University,
1-3 Machikaneyama Toyonaka, Osaka 560-8531, Japan

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, this technology has been extended to real-time observation of fluorescent dye-labeled molecules in living cells. Total internal reflection fluorescence microscopy is the major technique for this purpose. Using this technique, we have studied the process of early signal transduction of epidermal growth factor (EGF) in single molecules: binding of EGF to its receptor (EGFR) on the cell surface, dimerization of EGFR induced by binding of EGF, fluctuation of the structure of EGFR clusters, activation of EGFR through tyrosine phosphorylations on its cytoplasmic domain, and recognition of activated EGFR by a cytoplasmic adaptor protein, Grb2. EGF induces intracellular calcium response, sometimes caused by less than one hundred EGF molecules. Single-molecule studies suggested that this highly sensitive response to EGF was due to the amplification of the EGFR signal using dynamic clustering, reorganization of the dimers, and lateral mobility of EGFR on the cell surface. Through these studies, single-molecule analysis has proven to be a powerful technology to analyze intracellular protein systems.

Keywords: adaptor protein, calcium response, phosphorylation, signal transduction, total internal reflection fluorescence microscopy

Source: J Pharmacol Sci 93, 253 – 258 (2003).


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