Fig. 3: Orthogonal dual-tether pulling assay to monitor tension propagation in cells.

A Bright-field microscopy (left) and confocal fluorescence microscopy (right) images of two orthogonal tethers pulled by two optically trapped beads from a HEK293T cell dyed with DiI-C12. B Schematic illustration of the orthogonal dual-tether assay to monitor membrane tension propagation. Bead 1 moves outwards the cell extending tether 1 and thereby increasing the local membrane tension. If the tension perturbation propagates along the cell membrane within the experimental timescale, bead 2 will start moving towards the cell in the direction perpendicular to that of bead 1. C, D Representative plots of the tether force (C) and the bead position (D) of the orthogonal dual-tether pulling assay conducted in HEPES buffer with 150 mM NaCl. The position of each bead was normalized to its movement amplitude. Within the experimental accuracy, bead 2 did not respond to the movement of bead 1. N = 19 cells from 4 independent experiments. E, F Representative plots of the tether force (E) and the bead position (F) of the orthogonal dual-tether pulling assay conducted in HEPES buffer with 50 mM NaCl. The position of each bead was normalized to its movement amplitude. The left panels in (Fig. 3 C–F) correspond to the monitoring of the response of bead 2 in the direction perpendicular to that of the movement of bead 1. The right panels correspond to monitoring of the response of bead 2 in the same direction as that of the movement of bead 1. Note that the response of the bead 2 at 50 mM was only in the direction perpendicular to that of the movement of the bead 1. N = 7 cells from 3 independent experiments. Source data are provided as a Source Data file.