Movement of plasma-membrane-associated clathrin spots along the microtubule cytoskeleton

The current understanding of the role of plasma-membrane-associated clathrin suggests that clathrin-coated pits form at the sites of activated receptors and then, following internalization, the clathrin coat is rapidly shed. Utilizing total internal reflection fluorescence microscopy (TIR-FM), we have documented linear lateral motion of cell-surface-associated dsRed-clathrin spots parallel to the plasma membrane. Clathrin spot motility was observed in multiple cell lines (MDCK, CHO, Cos-7 and HeLa). In MDCK cells dsRed-clathrin spots moved along linear pathways up to 4μm in length with rates of approximately 0.8μm/s. Spots did not generally undergo internalization during movement. The motion of these puncta was coincident with the microtubule cytoskeleton, and depolymerization of microtubules reduced spot motility over 10-fold. Over-expression of the microtubule-associated protein tau-EGFP decreased spot run length by 40% without affecting the rate of movement. Thus dsRed-clathrin puncta move along the microtubule cytoskeleton parallel to the cell surface.