Diffusion properties of clathrin on the surface of isolated mouse liver nuclei by the fluorescence recovery after photobleaching technique

Abstract

Clathrin labeled with eosin maleimide showed physicochemical properties similar to the native clathrin. The diffusion coefficients of clathrin protomers and cages measured at 20°C by the fluorescence recovery after photobleaching technique (FRAP) were found equal to (9 ± 1) · 10 −8cm 2·s −1 and (3 ± 0.4) · 10 −8cm 2 ·s −1, respectively. After incubation with isolated mouse liver nuclei suspended in an aqueous buffer, FRAP measurements showed that 78% of clathrin was immobilized on the nuclear surface. This immobile fraction might correspond to aggregates of molecules resembling coated pits. The mobile fraction had a diffusion coefficient of 2.5 · 10 −9cm 2·s −1 which was reduced seven times when the suspension medium of the nuclei contained 50% sucrose, showing that the aqueous phase exerted an important drag on the clathrin molecules motion diffusing on the nuclear surface.