Figure 1: SMFS using AFM.
From: Mechano-adaptive sensory mechanism of α-catenin under tension
(A) α-Catenin fragments: WT MI-MIII (residues 276–634), mutant (MT) MI-MIII (M319G and R326E), MI (residues 276–393), and MII–MIII (residues 385–634). (B) Loading conditions (a) and (b). Single α-catenin molecules were loaded by NanoWizard 3 BioAFM (JPK Instruments, Berlin, Germany). The piezo-actuator was moved upward by 300 nm at a constant speed of 500 nm/s (“Loading (a)” in this study, orange line). To analyze the mechanical behavior of WT MI-MIII after mechanical activation, we introduced a holding time of 1 s at 40 nm of constant piezo-moving length (“Loading (b),” orange (initial loading), magenta (holding), and purple (further loading)). (C) Force curve analysis. First, the force curves with saw-tooth patterns (state (i) to (iv), orange curve) caused by single-molecule behaviors were identified, with the rupture force and final stiffness in the last peak (state (iv)). Next, the offset length Loffset was determined by WLC-model fitting to the last peak (green curve) with the fixed final contour length Lcf (143.2 nm (WT/MT MI-MIII), 46.8 nm (MI), and 99.6 nm (MII–MIII)). Finally, we measured the contour length Lc and peak unfolding force Fu at each force peak with transition force ΔFT (state (ii) to (iii)) greater than the threshold ΔFth (Methods: “Force curve analysis”).
