Actin filaments facilitate two steps of endocytosis.

Recent reports have suggested a role for microtubules in the endocytic process and pointed out the role for actin filaments for the early steps of apical endocytosis in polarized epithelial cells. However, these studies do not address the respective contribution of these two types of filaments along the endocytic pathway. In addition, several studies failed to demonstrate the role for actin filaments in clathrin dependent endocytosis in non polarized cells. In the present study we have investigated the role for both filaments in the endocytosis of two ligands internalized via clathrin coated pits (transferrin and alpha 2-macroglobulin) in a mouse hepatoma cell line. By immunocytochemical analysis with confocal microscopy, and biochemical analysis using a temperature sensitive step at 18 degrees C, we have shown that actin filaments are involved in two steps of the degradative pathway, and that microtubules are required at a stage in between. Actin filaments increase first, the uptake of ligands and second, their delivery to the degradative compartment, whereas microtubules are required to maintain the distribution of the late endocytic compartment in its juxtanuclear position and facilitate the delivery of the ligands to the degradative compartment upstream of the actin filament requirement. Furthermore actin filaments facilitate the recycling of transferrin from the perinuclear region to the plasma membrane. Our data indicate for the first time the sequential involvement of actin filaments and microtubules along one intracellular membrane trafficking pathway.