Fig 1.
Amyloidogenesis of full-length Tau and its K18 and K18ΔK280 fragments: The mother generations (G0).
(A) A schematic representation of the protein constructs used in this study: FL Tau (2N4R) containing an N-terminal His tag, K18 and K18ΔK280. The acidic inserts (N1, N2), proline-rich regions (P1, P2, P3), and microtubule-binding repeats (R1, R2, R3, R4) are indicated. (B) Kinetics of the de novo amyloid assembly of FL Tau (red), K18 (blue), and K18ΔK280 (green curve) probed by fluorescence of ThT. Error bars reflect standard deviations calculated for several trajectories measured simultaneously. (C) The intensity-magnified scale for the plot represented in B. (D) ATR-FTIR spectra of insoluble aggregates collected after 72-hour-long incubations with polyglutamate compared with the spectrum of dry film of poly-Glu in the absence of Tau polypeptides. TEM micrographs of FL Tau fibrils (E), K18 fibrils (F) and K18ΔK280 fibrils (G) formed during 72-hour-long fibrillization.
Fig 2.
Formation of G1, G2 and G3 generations of Tau amyloid fibrils.
(A) A scheme of preparation of mother (G0) de novo formed fibrils from monomers of FL Tau, K18 and K18ΔK280, and daughter fibril generations of FL Tau (G1, G2 and G3) obtained through seeding of FL Tau monomers with various templates. Kinetics of the self-assembly of G1 (B), G2 (C), and G3 (D) fibrils probed by ThT fluorescence. Insets show the same plots with magnified intensity scales.
Fig 3.
TEM images of G0, G1, G2 and G3 generations of Tau amyloid fibrils.
Red arrowheads indicate morphological variations in the fibrils: paired straight structures formed by K18 and propagated on to G1 and G2 generations of FL Tau fibrils (panels B, E, H), or PHF-like structures formed by K18ΔK280 and propagated on to FL Tau fibrils (panels C, F). Representative images were chosen. Scale bar of 200 nm applies to all micrographs.
Fig 4.
Limited proteolysis of G1, G2 and G3 generations of Tau fibrils by trypsin.
SDS-PAGE analysis showing proteolytic patterns of enzymatically digested fibrils (formed during 72-h fibrillization). The lanes correspond to products of trypsin-digested G1..G3 daughter fibrils of FL Tau induced by homologous seeding (FL Tau generations) and cross-seeding (K18 / K18ΔK280 generations). M lane shows molecular weight marker.