Juxtacrine signaling systems require direct interaction between ligand and receptor molecules on apposing cell surfaces. Physical contact between ligand and receptor cells triggers the transduction of chemical signals. We describe the development of a method to recapitulate the cell-cell interface of a juxtacrine system. This platform is composed of: (1) fluorescently tagged ligand molecules tethered to a supported lipid bilayer and (2) receptor-eGFP expressing cells. Ligand and receptor molecules are observed in separate channels using total internal reflection fluorescence (TIRF) microscopy, where each channel is quantitated by comparing fluorescence signal with calibration standards. This method can be used to determine the stoichiometry of binding in live cells and can be widely utilized to study any juxtacrine system. This presentation will describe how this method was specifically applied to the Notch signaling pathway.

Quantitative fluorescence imaging of intermembrane ligand-receptor complexes

243rd National Meeting (2012)

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