Membrane wound healing at single cellular level Rehana Afrin, PhD, Masakazu Saito, PhD, Takahiro Watanabe-Nakayama, PhD, Atsushi Ikai, PhD  Nanomedicine: Nanotechnology, Biology and Medicine  Volume 13, Issue 7, Pages 2351-2357 (October 2017) DOI: 10.1016/j.nano.2017.07.011 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 (A) A simplified view of the cell emphasizing relation between cell membrane and stress fibers. 1: cell membrane; 2: cytosol; 3: nucleus; 4: focal adhesion containing paxillin, zyxin, integrin, talin, among others; 5: ventral stress fiber; 6: transverse stress fiber: 7: dorsal stress fiber; 8: substrate. (B) Fluorescence labeling strategy of phospholipid bilayer with Kusabira Orange (KO) (λex=548nm, λem=559nm) (a) and cortical acto-myosin layer and stress fibers with GFP tagged β-actin (λex=475nm, λem=505nm) (b). See text for details. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Hole creation and repair process. A selected cell before (A) and after hole creation (B). In panel B, a newly created hole is circled. Scale bar=20μm. (C) Time sequence of refilling of a hole with time in seconds after hole creation as given below each image. Scale bar = 2μm. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 (A) Plot of the area of five small holes in Figure 2 as a function of time after their creation. Ordinate: temporary area of each hole. Abscissa: Time in seconds after detachment of the enzyme coated AFM probe from the cell surface. (B) Conceptual scheme of resealing of a hole with time dependent advancement of phospholipids from the periphery from 1 to 6. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 (A) Slow growth non-spherical as well as spherical blebs within 6 min of hole creation. (B) A case of numerous bleb formation soon after the AFM probe touched the cell surface without hole creation. Scale bar=20μm. Arrows show the points where the AFM probe touched the cells. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Lateral scanning AFM operation on intra-cellular stress fibers. (A) The AFM tip laterally scanned along the white dotted line in the direction of arrow head. (B) After lateral scanning, fiber 2 was found partially damaged (fluorescence intensity is reduced) while fiber 3 was completely cut. (C) Fiber 2 is repaired while fiber 3 retracted to 1/3 of its original length. Scale bar=20μm. The figure was reproduced from Hakari et al13 with permission. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Graphical representation of the increase/decrease of specific fluorescence to paxillin (top) and zyxin (bottom). The figure was reproduced from Watanabe-Nakayama et al12 with permission. Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2017 13, 2351-2357DOI: (10.1016/j.nano.2017.07.011) Copyright © 2017 Elsevier Inc. Terms and Conditions