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October 20, 2006

Shedding light on reactions in living cells

Researchers use single fluorescent molecules to reveal the details of biological interactions

Figure 1: A graphic representation of the tethered and extended forms of the EGFR, the predimer complex (top), and the binding of the two EGF molecules involving the formation of an intermediate (bottom).

EMBO/European Molecular Biology Organization/25/4220 (2006)

A team led by molecular biologist from RIKEN’s Discovery Research Institute has used a technique of tracking of single fluorescent molecules to unravel details of a key reaction in the control of proliferation, differentiation and migration of cells. The work demonstrated the application of such single-molecule visualisation and analysis to biological reactions in living cells.

The reaction the researchers studied—the binding of epidermal growth factor (EGF) with its receptor (EGFR) on the external cellular membrane—sets off a cascade of molecular events inside the cell. These events are important to the development of organs and tissues, but can initiate cancer. Until now, just how the reaction takes place has been unclear.

Cells sensitive to EGF usually have tens of thousands of EGFRs on their external membrane, yet measurable responses can occur when less than one per cent of these receptors are bound to EGF molecules. Recent crystallographic studies showed that the EGFR occurs in two forms—one of them folded up or ‘tethered’, the other stretched out or ‘extended’ (Fig. 1). It is known that EGF only triggers cellular activity only when it forms a complex of two EGF molecules and two EGFRs joined together. Two EGFRs can join together independently to form the basis of this complex.

The researchers added EGF molecules with attached rhodamine dye to sensitive cells. Fluorescent spots appeared on the cell surface where these molecules bound to EGFRs. Some of the fluorescent spots later increased in intensity by about twofold. After emitting constant light for a time, the spots then disappeared. Some spots disappeared in a two-stage process.

From their observations, which are reported in The EMBO Journal1, the researchers were able to calculate the rates of binding of EGF molecules to their receptors. These were tested against mathematical predictions arising from different binding reaction mechanisms. The closest fit was with the mechanism graphically represented in Figure 1, whereby two receptors in the extended conformation interact to form a single ‘predimer’ complex capable of binding two EGF molecules. After the first EGF molecule binds, the complex rearranges its shape to facilitate the binding of the second molecule. This intermediate form was predicted by the faster rate at which the second molecule binds.

“We are now using single molecule visualization to analyse the reactions inside cells downstream of the EGFR,” says project leader Yasushi Sako. “In future, this technique can be used in diagnosis and drug screening.”

References

  1. Teramura, Y., Ichinose, J., Takagi, H., Nishida K., Yanagida, T. & Sako, Y. Single-molecule analysis of epidermal growth factor binding on the surface of living cells. The EMBO Journal 25, 4215–4222 (2006). || (Link)