Fig. 5: Amylin fibril disruption and degradation using designed binder.
From: Diffusing protein binders to intrinsically disordered proteins
a,b, NS-EM visualization of fibril dissociation by amylin-36αβ at both elongation (a) and mature (b) phases. Scale bars, 100 nm. c, The ThT assay revealed that all four binders could strongly inhibit fibril formation at a binder:amylin molar ratio of 1:4. d, Amylin-36αβ could dissociate fibrils at the elongation phase in a concentration-dependent manner. Amylin-36αβ was added at 3 h during the elongation phase of amylin fibrils (as marked by the dashed line), and the ThT assay was performed to monitor the process. The red and blue dots indicate amylin-36αβ:amylin molar ratios of 1:4 (10 μM binder) and 1:40 (1 μM binder), respectively. e, The ThT assay was performed after the mature amylin fibrils were formed for 24 h and, at the same time, amylin-36αβ was added. The red and blue dots indicate that the amylin-36αβ:amylin molar ratios are 1:4 (10 μM binder) and 1:40 (1 μM binder), respectively. f,g, Confocal microscopy images of Hep3B cells treated with Alexa Fluor 647-labelled amylin fibrils (f; 24 h incubation at 37 °C) or monomers (g), in complex with amylin-36 (top), amylin-36–ASGPR (middle) or amylin-36–IGF2R (bottom). DAPI (blue) stains nuclei, LAMP1 (green) marks lysosomes, and amylin (magenta) is visualized via fluorophore-conjugated streptavidin. Composite panels (rightmost) show merged channels. Scale bars, 25 μm (f,g). h, Quantification of internalized amylin fibrils (left) and monomers (right) by flow cytometry; the median fluorescence intensity (MFI) was measured. Data are shown as mean ± s.d. from three biological replicates.
