Extended Data Fig. 10: Optical tweezer calibrations for pulling and connection-breaking assays.
a, Schematic representation of the optical tweezers binding assay. Nanobody-beads were held for 3 s on the cell surface ∼ 1 µm behind the tip of the lamellipodium using the laser trap. Beads were scored as bound if they remained focused in the plane of the membrane for more than 10 s after turning the laser tweezers off. b, Binding fractions as a function of the Nanobody-Biotin/BSA-Biotin ratio on beads for GFP-β3-integrin (n0/1 = 24 cells, n1/2 = 11 cells and n1/0 = 43 cells, pooled over 8, 3 and 15 independent experiments), TM-Nap1 (n0/1 = 6 cells, n1/2 = 46 cells and n1/2 = 29 cells, pooled over 2, 9 and 5 experiments) and TM-ctr (n0/1 = 3 cells, n1/2 = 12 cells and n1/0 = 5 cells, pooled over 1, 7 and 2 experiments), (mean ± s.e.m. for cells). c, Fractions of pulled beads below 1 pN (light histograms), between 1 pN-2 pN (mid shaded histograms) and above 2 pN (dark histograms), for TM-Nap1 (n = 5 experiments), TM-Sra1 (n = 3 experiments), TM-Sra1-WCA* (n = 3 experiments), TM-Sra1-AD (n = 3 experiments) and TM-ctr (n = 4 experiments), (mean ± s.e.m. for experiments). d, Pulled fraction as function of the maximal escapement force determined for defined trap stiffnesses. TM-Nap1 (n = 329 pulling assay), TM-Sra1 (n = 399 pulling assay), TM-Sra1-WCA* (n = 372 pulling assay), TM-Sra1-AD (n = 211 pulling assay) and TM-ctr (n = 157 pulling assay). c, d, Data pooled from independent experiments: TM-Nap1 (pooled over 28 cells from 5 experiments), TM-Sra1(pooled over 11 cells from 3 experiments), TM-Sra1-WCA* (pooled over 12 cells from 3 experiments), TM-Sra1-AD (pooled over 18 cells from 3 experiments) and TM-ctr (pooled over 9 cells from 4 experiments). e, Fractions of breaking forces bellow 1 pN (dark histograms), between 1 pN-2 pN (mid shaded histograms) and above 2 pN (light shaded histograms). TM-Nap1 (11 cells), TM-Sra1(9 cells), TM-Sra1-WCA* (22 cells), TM-Sra1-AD (4 cells) and TM-ctr (5 cells), (mean ± s.e.m. for cells). f, Distributions of breaking forces for nanobody-beads bound to TM-Nap1 (n = 54 breaking events), TM-Sra1 (n = 65 breaking events), TM-Sra1-WCA* (n = 218 breaking events), TM-Sra1-AD (n = 8 breaking events) and TM-ctr (n = 16 breaking events). e, f, Data pooled from independent experiments: TM-Nap1 (pooled over 11 cells from 5 experiments), TM-Sra1 (pooled over 9 cells from 5 experiments), TM-Sra1-WCA* (pooled over 22 cells from 8 experiments), TM-Sra1-AD (pooled over 4 cells from 3 experiments) and TM-ctr (pooled over 5 cells from 3 experiments). g, Using a piezoelectric motor, the sample was moved at increasing velocities. The increasing viscous drag force exerted on the bead triggered larger displacements until escapement from laser trap (left). Displacement versus time plots from the trap center as function of the viscous drag force at different laser powers (2.4, 3.9 and 7.9 mW) (right). h, Displacement amplitudes as function of the viscous drag force. Trap stiffnesses were calculated for different laser powers, and corresponded to the slope of displacement versus force curves. i, Trap stiffness as a function of laser power. j, Maximal trap force as function of laser power. Also see Supplementary Table 4. Numerical source data are provided in Source data.
