Single-molecule visualization or cell signaling processes in living cells

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Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, we extended this technology to real-time observation of fluorescent dye-labeled molecules in living cells. Using this technique, the process of dimerizalion, a key step of EGFR signal transduction was observed. EGERs made dynamic and transient clusters on the cell surface even in the absence of EGF. In most cases (ca.90%) dimers of EGF/EGFR complexes were formed by the direct binding of EGF molecules in solution to the clusters of EGFR. This clustering should be important to biological function of EGFR since disruption of the actin cytoskeleton resulted in dispersion of EGFR clusters and decreased tyrosine phosphorylation. Phosphorylation of EGFR was visualized one by one in membrane-permeabilized cells using fluorescensly labeled Fab' fragment of an antibody specifically recognizes activated EGFR. The number of activated EGFRs often became lager than that of bound EGFs by a factor of 5-10. Clustering and lateral movement of EGFR also seem to be important for this signal amplification process. In addition so EGFR, single-molecules of nerve growth factor, small G proteins, Raf1 kinase, and a fluorescent analogue of cAMP were observed in living cells. Single-molecule imaging enables us to observe localization, movement, oligomerization and turnover of individual signaling molecules in the plasma membrane of living cells.
日本組織細胞化学会の論文

Single-molecule visualization or cell signaling processes in living cells

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