Figure 1.
Annular oligomers along the TTR assembly pathway. A.
2×2 µm AFM scan displaying TTR oligomers some of which form short (<100 nm) linear structures. B. 500×500 nm AFM scan of the square area marked in Figure 1.A, indicating that the sample is almost exclusively populated by annular oligomers. In the center, annular oligomers are associated to form a linear aggregate. C. Height contrast image of an annular TTR oligomer. Octameric symmetry is indicated with numbers referring to component monomers. The circle and line mark the perimeter and diameter, respectively, across which topographical height data were measured and plotted. Scale bar, 10 nm. D. Topographical height profiles of the annular oligomer taken along its perimeter (black) and its diameter (inset, red). E. Distribution of the topographical height of annular oligomers. “n” represents the number of height data points for 57 annuli. F. Distribution of peak-to-peak periodicity along the annulus perimeter (n = 106; 33 annuli).
Figure 2.
Formation and disappearance of annular oligomers.
A. Dynamic light scattering spectra of native TTR and at given time points after the start of acidification where the emergence of a small population of larger particles follows a trend towards smaller apparent hydrodynamic radii (Rhapp). B. Time course of the apparent size of the different populations during aggregation and their corresponding weighted average. The arrows indicate the times points where images shown in C and D were taken. C & D. AFM images (phase contrast) of particles taken at 9 and 12 h respectively and where annular oligomers (C) and spheroid (D) oligomers can be observed. The inset represents a 50×50 nm topography image of the corresponding samples (height scale up to 2.5 nm) E. Height-contrast AFM image of annular oligomers undergoing transitions. E. Magnified view of fusing annular oligomers indicated in D. Height, amplitude and phase contrast images (left to right) are shown. Scale bar, 10 nm.
Figure 3.
TTR spheroid oligomers and protofibrils.
A. 1×1 µm2 AFM height contrast image of a mixed population of spheroid oligomers and short protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. Inset, magnified view of a protofibril displaying a stack-like arrangement of flat, disc-shaped oligomers reminiscent of annular origin. B. 1×1 µm2 AFM height contrast image of a mixed population of spheroid oligomers and longer protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. Inset, magnified view of a protofibril in which the underlying periodic structure is probably helical. C. Topographical molecular volume histogram of 341 (n) spheroid TTR oligomers. The numbers above the modes correspond to the mean values of gaussian fits.
Figure 4.
Disassembly of TTR protofibrils.
A. AFM height contrast image recorded after 1 minute of sample dilution into PBS. B. AFM height (top) and amplitude (bottom) contrast images recorded after 5 minutes of sample dilution into PBS. C. AFM height (top) and amplitude (bottom) contrast images recorded after 15 minutes of sample dilution into PBS. Insets, magnified image of a single annular oligomer (left) and a laterally-associated doublet of annular oligomers (right). D. Distribution of the diameter of annular oligomers observed during assembly (yellow) and disassembly (purple). E. Distribution of the topographical height of annular oligomers observed during assembly (yellow) and disassembly (purple).
Figure 5.
Model of TTR protofibril assembly and disassembly.
Relevant dimensions and periodicity parameters of the intermediates are indicated where applicable. Length of the arrows scale with the hypothesized transition kinetics.