Figure 1.
Mutant SOD1:YFP fusion proteins form obvious intracellular inclusions.
As described in Methods, CHO cells were transiently transfected with expression plasmids for WT or A4V SOD1 fused to YFP. After 24 or 48 hours the cells were fixed with 4% paraformaldehyde and then images were captured to assess the frequency of inclusion formation. Cells expressing the A4V SOD:YFP fusion protein contain inclusion-like structures at a high frequency.
Table 1.
Aggregation of SOD1 mutants in transfected cells.
Figure 2.
Mutation of cysteine 111 to serine or tyrosine differentially affects inclusion formation.
CHO cells were transiently transfected with expression plasmids for each mutant and imaged 24 or 48 hours later as described in Figure 1. Mutation of cysteine 111 to serine does not induce inclusion formation whereas the disease associated mutation of cysteine to tyrosine produces inclusion-like structures at a high frequency. For comparison, cells expressing WT SOD1:YFP fusion proteins are also shown.
Figure 3.
Simultaneous mutation of cysteines 6 and 111.
CHO cells were transiently transfected with expression plasmids for each mutant and imaged 24 or 48 hours later as described in Figure 1. For the C6G-C111S and C6G-C111Y mutants (Panels A–D), the rate of inclusion formation was slower than that of the positive control mutant A4V (Panels E and F). At 48 hours, the frequency of inclusion-like structures in cells expressing the double mutant C6G-C111S remained low compared to C6G-C111Y (see Table 1).
Figure 4.
Simultaneous mutation of all four cysteines in SOD1.
CHO cells were transiently transfected with expression plasmids for each mutant and imaged 24 or 48 hours later as described in Figure 1. Mutants lacking cysteine formed obvious inclusion-like structures. FSYR; C6F/C57S/C111Y/C146R. GSYR; C6G/C57S/C111Y/C146R.