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, that TβRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-β1stimulated cells. This strongly supported our former discovery that TβRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TβRII molecules. On the basis of these observations, we proposed a mechanism of TGF-β1-induced TβRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling