The biological homogeneity of NM fibrils was confirmed by protein-only transformation. (a) Diagrams of wild-type Sup35. The black segments in the N-terminal region of Sup35 (N) represent oligopeptide repeats. The highly charged nature of the middle region (M) is indicated. The C-terminal region (C) functions in translation termination. (b–f) Biologically distinct prion states were induced when prion-minus cells were transformed with fluorescently labeled fibrils formed under different conditions. (b) Colonies transformed with buffer alone. (c) Colonies transformed with spontaneously assembled NM fibrils. A mixture of prion phenotypes is highlighted in the blue box. (d) Colonies after “protein-only” transformation at 4°C with NM fibrils that were seeded by lysates of yeast cells that carried a strong prion element. The uniform strong prion phenotypes are indicated by the light pink-colored colonies in the blue box. (e) Colonies after “protein-only” transformation at 37°C with NM fibrils that were seeded by lysates from cells carrying a weak prion element. The uniform weak prion phenotypes are indicated by the dark salmon-colored colonies in the blue box. (f) Quantification of transformation efficiency and specificity of distinct prion fibrils. Strong fibrils refer to the fibrils used in (d), while weak fibrils refer to the fibrils used in (e).

NM monomers effectively and specifically bind individual NM fibrils to a glass surface on one end and a polystyrene bead on the other. (a) Schematic diagram of the method for the attachment of NM fibrils. Monomers (blue) are adhered to the glass surface and the remaining surface is coated with casein (light green). Green circles, fluorescently labeled NM monomers; white circles, wild-type NM monomers. Polystyrene beads (light blue circles) labeled with Alexa488 (green triangle) and Alexa555-NM (blue) are introduced and captured by the other free ends of the fibrils. (b) Top, The molecular architectures and amino-acid compositions of Saccharomyces cerevisiae NM (blue) and Candida albicans NM (orange). Bottom, schematic illustration of the experiment demonstrating the specificity between pre-bound monomers and fibrils. From left to right, deposition of Candida NM on the glass surface; incubation of Saccharomyces NM fibrils in the flow channel; imaged after washing. (c) Schematic diagram of the detachment of NM fibrils from the glass surface with a treatment of TEV protease. The TEV recognition site is shown in red and highlighted in the inserts. The M domain (blue line) binds with the glass surface and the N domain (blue circle) interacts with the end of NM fibrils. The bead was held in position by the optical trapping (light pink).

Stretching NM fibrils by optical trapping at high forces with simultaneous fluorescent imaging. (a) Schematic of the trapping instrument with dual trapping capabilities used for high force experiments (see Methods for details). 1024, nm, trapping laser; 975 nm, position detection laser; 532 nm, fluorescence imaging. (b) Schematic representation and fluorescence snapshots of the experiments. From left to right, the bead was first centered on top of the attachment point, and then the stage was moved along the x-axis until the fibril was fully extended. (c) Representative pulling and relaxation traces for NM fibrils in the presence of normal assembly buffer. The force and the position of the stage were plotted as a function of time. Black line, the stretching and holding phase; red line, the relaxation phase; blue line, position of the bead relative to the attachment point of the tether on the glass surface. (d) Left, force applied vs. fibril extension length (force-extension curve) for the tethered fibril experiencing one stretching-relaxation cycle. Black line, stretching and holding phase; red line, relaxation phase. The x-axis is expanded to illustrate the behavior in the fully extended position. Right, the force-extension curves for a single tethered fibril that experienced four sequential stretching-relaxation cycles superimpose upon each other. For clarity, the holding phase is not shown. Stretching and relaxation curves from the same cycle are shown in the same color, with subsequent curves in a different color.

Representative pulling traces (force vs. time) for NM fibrils under different experimental condition. (a) Force-extension curves of NM fibrils in the presence of 1.2 M GdHCl. Black arrows highlight sudden force changes in the stretching and holding traces. Red arrows mark rupture of the tethered fibril with a concomitant drop to zero force. (b) Representative force-extension curves for two NM fibrils that experienced multiple stretching-holding-relaxation cycles in the presence of 1.2 M GdHCl. Black, light blue, blue, light pink and green correspond to cycles 1–5 for each fibril. For clarity, only stretching phases are shown. (c) Force-extension curves in the presence of 50 μM DAPH. (d) The structures of DAPH and SA.

Expansion or deletion of the repeat region dramatically changed mechanical behavior of prion fibrils. (a) A previously proposed β-helix model for NM fibrils. The M domain is not involved in the amyloid core and is not shown. The color scheme for the N domain is demonstrated on the top left. An expansion of two monomers is shown on the top right. (b) and (c), top, schematic illustration of R2E2 and RΔ2-5. The blue segments in the N-terminal region represent oligopeptide repeats. Middle, R2E2 (left) and RΔ2-5 (right) fibrils imaged by transmission electron microscopy. Scale bar, 100 nm. The R2E2 fibrils have an average diameter of 16 ± 2 nm, slightly wider than that of wild-type “strong” NM fibrils, 12±2 nm. The RΔ2-5 fibrils have a diameter comparable to wild-type fibrils, but many showed a distinct wavy morphology. Bottom, protein-only transformation of R2E2 (left) and RΔ2-5 (right) fibrils. (c) Representative force-extension curves for R2E2 (left) and RΔ2-5 (right) fibrils. The black traces, stretching phase; the red traces, relaxation phase. Black arrows, force-dropping events in the stretching curves; green arrows, force plateaus in the relaxation curves at low forces. Insert, expansion at the low force regime. The stretching and relaxation curves were fit to a modified WLC model, shown as black and red curves, respectively, in the inserts. The deviation of the relaxation curves from the WLC fitting is highlighted by the green arrow.

The distribution of the length of extension is broad. (a) Left, An example of WLC fits used to determine the length added to the fibril at each force-dropping event that occurred during the stretching phase. Right, a linear correlation was obtained between the length of extension determined from WLC fits and the instantaneous bead displacement that occurred upon the unfolding. Black Points, length of extension from WLC fits to wild-type NM fibrils; gray points, length of extension from WLC fits to RΔ2-5 fibrils. (b) Histogram of the length of extension for NM fibrils at each force- dropping event in the presence of 1.2 M GdHCl (left) or 50 μM DAPH (right). (c) Histogram of the extension lengths obtained with RΔ2-5 fibrils. A 12nm and a 24nm bin width were used for (b) and (c) respectively (Supplementary methods). (d) Rupture time vs. force for the RΔ2-5 fibrils. The rupture events that occurred during the holding phase were fit to a Bell model as described in the methods.

Cartoon representation of the mechanical unfolding of NM amyloid fibrils with different structures. (a) A possible mechanism with the β-pleated sheet model. (b) A possible mechanism with the β-helix model. See discussion for details.