Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface

Single green fluorescent protein (GFP) molecules were successfully imaged f or the first time in living cells. GFP linked to the cytoplasmic carboxyl t erminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells , and observed using an objective-type total internal reflection fluorescen ce microscope. Based on the fluorescence intensity of individual fluorescen t spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, s uggesting that oligomerization of E-cadherin takes place before its assembl y at cell-cell adhesion sites. The translational diffusion coefficient of E -cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because s uch large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapp ing model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the m embrane skeleton on oligomers (compared with monomers). The presence of man y oligomers greater than dimers on the free surface suggests that these gre ater oligomers are the basic building blocks for the two-dimensional cell a dhesion structures (adherens junctions).