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. 2017 Oct 1;28(20):2650–2660. doi: 10.1091/mbc.E17-01-0080

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© 2017 Ng et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Local force application by the tip of an atomic force microscope cantilever is sufficient to induce transcellular tunnels in endothelial cells. (A) Schematic diagram of the experimental setup. Using the AFM, a tipped cantilever is extended toward a spread HUVEC until the tip is in contact with the cell at 5 nN force. Then the applied force is slowly increased at a rate of 1 nN/s until a tunnel is observed by TIRF imaging of the cell membrane or the tip has been extended by 3–5 µm. The cantilever is retracted while the progression of the opening and closing of the tunnel is tracked by time-lapse imaging. (B) Time-lapse TIRF images of a HUVEC forming a tunnel when indented by the tip of an AFM cantilever. The cell was expressing GFP-MIM-I-BAR and LifeAct-mCherry and labeled with the CellMask far red membrane dye. Opening of the tunnel was followed by localization of MIM around the aperture and actin-rich wave formation to close the aperture. The arrowheads indicate location of the AFM cantilever tip. The time points of each image relative to tip contact (t = 0 s) are displayed above the image. Scale bar: 10 µm. (C) Example force trace during an indentation experiment. (D) Kymograph of a region of the cell in A, outlined by the yellow box, showing tunnel opening at a higher temporal resolution of 2 s intervals. The yellow dotted line marks the location of the tip and the yellow solid lines trace the opening of the tunnel. (E) Trace of AFM tip distance from the glass substrate, which corresponds to the force trace in C. This trace also shows the change in cell height as the tip continued to deform the cell at higher forces. The arrows in C–E indicate the point when the tunnel opened. Inset shows tip distance trace when set occurred. (F) Example force vs. tip-substrate distance trace from a different cell, showing a gradual increase in force at the beginning of indentation, followed by a steeper increase in force as a function of deformation. A step displacement of the tip occurred at constant force due to full penetration of the cell when a tunnel opens.