Stepwise dynamics of epitaxially growing single amyloid fibrils

Abstract

The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid beta25-35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm(-1)) growth phases that extended the fibril by approximately 7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid beta1-42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.

Fig. 1.

Scanning-force kymography. (a) Schematics of the method. Rapid scanning is along the fibril axis, and the perpendicular slow scan is disabled. (b) Acquisition of a scanning-force kymogram. The 2D image is shifted to the kymogram upon disabling the slow scan (capped arrow). Rapid scan direction is horizontal. Grayscale intensity corresponds to topographical height. (c) Kymogram (Upper) converted to a time-dependent growth–distance plot (Lower). White and black arrowheads mark corresponding small steps. (Inset) Example of sigmoid fit onto a growth step.

Fig. 2.

Quantitative analysis of discontinuous amyloid fibril growth. (a) Distribution of pause time measured on the fast-growing end. Pauses are considered if they last for at least three consecutive line scans (≈1 s). The continuous line is the exponential fit. (Inset) Distribution of step size for the fast- and slow-growing fibril ends. Gaussian fits to the peaks in the fast-growing-end histogram are at 6.5 ± 0.2 nm, 13.3 ± 0.3 nm, 23.2 ± 0.3 nm, 32.5 ± 0.4 nm, and 40 ± 0.6 nm, respectively. (b) Step rate versus step time for the total number of observations (including fast and slow ends, n = 372). (Upper Inset) Step time versus total peptide concentration. (Lower Inset) Step rate versus total peptide concentration. (c) Kymograms displaying cooperative run-on events by the coalescence of ≈7-nm steps.

Fig. 3.

Model of amyloid assembly dynamics. (a) Schematic diagram of fibril-end kinetics. k₁ and k₂ are first-order rate constants of the transition between the blocked and growing states and vice versa, respectively. k_ON and k_OFF are second-order rate constants of rapid growth and depolymerization, respectively. (b) Hypothetic energy landscape of epitaxial amyloid fibril assembly. (c) Concentration-dependent kymograms of Aβ25–35 fibrils, demonstrating stepwise disassembly events. Gray and white block arrows indicate assembly and disassembly directions, respectively. Black arrowheads indicate backstep events. (d) Kymogram of Aβ1–42 on HOPG, revealing net stepwise disassembly. Grayscale intensity corresponds to the phase shift of the sinusoidally oscillated cantilever. White arrowheads indicate small assembly steps.