Involvement of Actin Dynamics in Endocytotic Process Revealed by Fast-Scanning Atomic Force Microscope

The endocytosis proceeds with a series of morphological changes of the plasma membrane, which is mediated by a successive assembly of a number of membrane-associated proteins. In addition to a local binding of those proteins, a mechanical tension of the membrane, as well as a dynamics of cortical cytoskeletal network is also involved in this process. Here, we utilized a fast-scanning atomic force microscope (AFM) which is specially designed for live-cell imaging (BIXAM), to directly visualize the dynamic morphological changes of the plasma membrane and cortical actin network, together with localizations of fluorescently-labeled proteins. COS-7 cells expressing EGFP-fused clathrin were observed by BIXAM. Overlaying confocal fluorescence images and AFM images clearly identified clathrin-coated membrane pits on the cell surface. The diameter of the pit was ∼300 nm, which is similar to that observed by an electron microscope, and the life time was 300 seconds. The membrane started to invaginate 10-20 seconds after the initial accumulation of clathrin. The clathrin signal kept slowly increasing, and suddenly disappeared 20-30 seconds after the pit closes. Interestingly, when the pit closes, a brief swelling of the adjacent membrane (∼50 seconds, ∼50 nm in height) appeared and covered the pit. Knock-down of dynamin did not affect this closing motion, indicating that dynamin is not necessary for the pit closure in COS-7 cells. Scanning of cortical actin with increased loading force of AFM, we found that the membrane swelling accompanied with local formation of densed cortical actin network. Inhibitors for actin polymerization/depolymerization (cytochalasin B and Jasplakinolide), as well as inhibitor for Arp2/3 complex (CK-666), inhibited the formation of the membrane swelling and caused reversible open-close motions of the pit, suggesting that actin dynamics is a major driving force for irreversible closing of the clathrin-dependent membrane pit.