Figure 4. Mechanical stress induces the formation of ThT-positive fibrils of polyubiquitin chains.

(a) EM images of sheared samples: linear diubiquitin (left upper), linear hexaubiquitin (left lower), K48-linked diubiquitin (middle upper), K48-linked hexaubiquitin (middle lower), K63-linked diubiquitin (right upper) and K63-linked hexaubiquitin (right lower) show that all of these types of polyubiquitin form fibrils with amyloid-like morphology when subjected to mechanical stress. The shear stress was applied as an agitation at a rotational speed of 25 or 33 s⁻¹. Scale bar, 100 nm. (b) Thioflavin T fluorescence emission spectra of sheared monoubiquitin (left, green), linear diubiquitin (left, black), linear hexaubiquitin (left, red), K48-linked diubiquitin (right, orange) and K63-linked diubiquitin (right, purple). Shear stress was applied as an agitation at a rotational speed of 25 s⁻¹ for 90 h. For monoubiquitin and K48-linked diubiquitin, the applied shear stress was that of 33 s⁻¹ for 90 h. (c) Fibril formation of sheared K63-linked diubiquitin (purple), linear diubiquitin (black) and hexaubiquitin (red), as followed by ThT fluorescence. K48-linked diubiquitin (orange) and monoubiquitin (green) did not show relevant increases in fluorescence. Shear stress was applied by agitation at a rotational speed of 25 s⁻¹. (d) Comparative analysis of the kinetics of fibril formation by polyubiquitin chains of different linkage type and chain length with transition temperature. Rate constants were obtained by fitting the data to first-order kinetics. The values represent the average of two independent experiments. Error bars, the standard error of the mean. *P<0.05 (Student’s t-test).