IMAGING THE LAMELLIPODIUM OF MIGRATING EPITHELIAL-CELLS IN-VIVO BY ATOMIC-FORCE MICROSCOPY

Cell locomotion originates at a specific region of the cell surface, the leading edge of a migrating cell. Various factors have been proposed to contribute to the propulsion of a cell over the substratum. Rapid turnover processes of cytoskeletal elements inside the cell and insertion of new plasma membrane at the leading edge of the cell permit the extension of a cell in a given direction. Our goal was to image in vivo plasma membrane turnover by means of atomic force microscopy (AFM) and to resolve dynamic processes at the nanometer level. As an experimental model we used migrating kidney cells derived from the Madin-Darby canine kidney (MDCK) cell line that was transformed by alkaline stress. These so-called MDCK-F cells exhibit spontaneous calcium-dependent oscillatory activity of plasma membrane potential associated with cell locomotion. We imaged cells during migration and observed dynamic invagination processes in the cell surface close to the leading edge, indicating internalization of plasma membrane. Invaginations were prevented by removal of;calcium from the perfusate. During calcium reduction plasma membrane uncoupled from the underlying cytoskeleton and lipidic pores with diameters of about 30 nm could be disclosed and imaged. This study demonstrates that the AFM can readily trace dynamic physiological processes in vivo, emphasizing the potential role of calcium in maintaining plasma membrane integrity and function.