ACTIN-FILAMENTS FACILITATE 2 STEPS OF ENDOCYTOSIS

Recent reports have suggested a role for microtubules ire the endocyti c process and pointed out the role for actin filaments for the early s teps of apical endocytosis in polarized epithelial cells. However, the se studies do not address the respective contribution of these two typ es of filaments along the endocytic pathway, In addition, several stud ies failed to demonstrate the role for actin filaments in clathrin dep endent endocytosis in non polarized cells, In the present study we hav e investigated the role for both filaments in the endocytosis of two l igands internalized via clathrin coated pits (transferrin and alpha 2- macroglobulin) in a mouse hepatoma cell line, By immunocytochemical an alysis with confocal microscopy, and biochemical analysis using a temp erature sensitive step at 18 degrees C, we have shown that actin filam ents are involved in two steps of the degradative pathway, and that mi crotubules are required at a stage in between, Actin filaments increas e first, the uptake of ligands and second, their delivery to the degra dative compartment, whereas microtubules are required to maintain the distribution of the late endocytic compartment in its juxtanuclear pos ition and facilitate the delivery of the ligands to the degradative co mpartment upstream of the actin filament requirement. Furthermore acti n filaments facilitate the recycling of transferrin from the perinucle ar 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.