Stretching, Unfolding, and Deforming Protein Filaments Adsorbed at Solid-Liquid Interfaces Using the Tip of an Atomic-Force Microscope

Cells move by actively remodeling a dense network of protein filaments. Here we analyze the force response of various filaments in a simplified experimental setup, where single filaments are moved with an atomic-force microscope (AFM) tip against surface friction, with the AFM operating in the torsional mode. Our experimental findings are well explained within a simple model based on Newtonian mechanics: we observe force plateaus, which are the signature of the sequential stretching of single repeat units, followed ultimately by deformation of the whole polymer shape.

Figure 1

Manipulation of collagen fibrils adsorbed to mica, pretreated with 1 M ${\mathrm{MgCl}}_2$ and immersed in PBS. Panels (a) and (b) were imaged prior to and after manipulation, respectively. Arrowheads identify two manipulations yielding a bent fibril with a cusped shape (1) and a stretched and broken bundle of fibrils (2).

Figure 2

(a) AFM image of single wild-type desmin filaments obtained after manipulation (adsorbed to mica and immersed in 25 mM Tris-HCl, 100 mM NaCl, $p\mathrm{H}$ 7.5). (b) Shape of a DC-WLC manipulated by a point probe at its middle for two cantilever displacements. (Dots represent beads and lines represent linkers.) Parameters: $v=50\mathrm{nm}/\mathrm{s}$ (as in experiment), $L_p=0.4\mathrm{nm}$, $L_c=3200\mathrm{nm}$, $L_0=1000\mathrm{nm}$, $N=25$ beads, and $N\nu =0.3\mathrm{N}\mathrm{s}/\mathrm{m}$. (c) Force versus displacement curves corresponding to panel A. Theoretical fit (broken line): $L_c=2200\mathrm{nm}$, $N=20$, others as in panel (b). (d) Force versus displacement curves of desmin filaments containing an equal amount of wild-type proteins and mutant D399Y proteins [9] with theoretical fits. Arrowheads indicate tip displacements corresponding to the shapes presented in panel (b). Parameters (dotted curve): as in panel B; broken curve: $L_p=2\mathrm{nm}$, $L_c=1600\mathrm{nm}$.

Figure 3

Theoretical and experimental models. (a) Shapes of a DC-WLC chain manipulated by a point probe. Parameters: $L_p=1\mathrm{nm}$, $L_c=1200\mathrm{nm}$, $N=100$, $N\nu =1\mathrm{N}\mathrm{s}/\mathrm{m}$, others as in Fig. 2b. Inset: Schematic of the DC-WLC. (b) Force versus displacement curves for the same chain manipulated at its middle (solid line) and at one end (broken line). (c) AFM image of a collagen fibril after manipulation revealing a similar cusp shape. (d) A 12 cm long rubber band adsorbed to a glass plate coated with silicon grease (Corning) and manipulated at an average velocity of $3\mathrm{mm}/\mathrm{s}$. (e) Force versus displacement curves for two DC-WLC chains, with $L_c=1200$ and 2000 nm for solid, dashed curves, respectively. [$N=20$ beads; other parameters as in panel (a).]

Figure 4

(a) Comparison of the DC-WLC fit of Fig. 2d (dotted line) with a two-state model fit (broken line) for the same experimental curve. (b) equilibrium stretching curves for the two models: two-state (solid line) and DC-WLC (dotted line).