Membrane wound healing at single cellular level

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A₂ was made to contact the cell membrane. A small dark hole (4-14 μm² in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014μm²s^-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).