Figure 2

The BiFC assay reliably captures the differences between wild-type and mutant SOD1. (A) Schematic illustration of the DNA constructs of the human SOD1 fused to truncated Venus fluorescent protein segments using a larger N-terminal fragment of Venus (V_N), corresponding to amino acids 1–158, and a smaller C-terminal fragment (V_C), corresponding to amino acids 159–239. (B) Schematic presentation of BiFC assay. Dimerization of WT SOD1 fused to Venus fragments generates fluorescence from the fusion of V_N and V_C fragments. (C) In the A4V SOD1 mutant, Venus fluorescence complementation allows to detect amyloid-like filament formation (aggregates/inclusion) due to non-native (unstable) A4V SOD1 dimerization. Representative images showing the different negative and positive controls to demonstrate the ability of BiFC assay to differentiate between stable, unstable or no SOD1 dimerization. Reciprocal negative controls of SOD1 V_C with empty V_N (V_N + SOD1 V_C or V_N SOD1 with empty V_C (V_C + V_N SOD1) vectors showing no fluorescence due to lack of dimerization in the absence of complimentary SOD1. Venus is pVenus N1 empty plasmid that does not contain SOD1 and only expresses VFP; C6S, a positive control, is an internal non-specific mutation and interaction of V_N C6S SOD1 and C6S SOD1 V_C presents uniform fluorescence WT represents the interaction of V_N WT SOD1 and WT SOD1 V_C displayingeven fluorescence A4V, aSOD1 mutant and represents the interaction of V_N A4V SOD1 and A4V SOD1 V_C leading to the aggregates/inclusion formation (shown by white arrows), scale bar: 25 μm. Left to right panel for each control shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 Vc or SOD1 V_N) and merged images respectively.