Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging

The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TβRII molecules on the plasma membrane, and then we recorded the individual newly appeared TβRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TβRII-GFP molecules were analyzed. We reported, for the first time...