Figure 6.

MM87 or M1 C448Y mutants do not affect MT-severing activity of WT M87. TRE/M87WT cells were used for transient transfections with pTRE/M87CY or pTRE/M1CY. Anti-spastin antibody was used to evaluate levels of spastin in TRE/M87WT cells cultured with or without Dox as well as in cells coexpressing WT and mutated spastin isoforms. The fluorescence intensity after staining with anti-tubulin antibody was used to quantify MT levels. A, No spastin expression and MT severing was detected in TRE/M87WT cells cultured without Dox. B, The 18 h exposure to 0.5 μg/ml Dox results in moderate WT M87 expression in >90% of TRE/M87WT cells (outlined in yellow). C, The level of MT-severing in cells coexpressing WT M87 and M87 C448Y (outlined in red) is indistinguishable from those in cells that express only WT M87 (outlined in yellow). D, MT severing was not diminished in cells coexpressing WT M87 and M1 C448Y (outlined in red). E, Compared with control nonexpressing cells, MT severing significantly lowered tubulin fluorescence intensity in cells expressing WT M87 alone or together with the mutant M87 or M1. Quantitative analysis of tubulin fluorescence intensity revealed comparable level of MT loss in cells expressing WT M87 alone or coexpressing WT M87 and M87 C448Y or M1 C448Y. That result indicates that mutated spastin isoforms do not affect severing activity of coexpressed WT spastin. Scale bar, 15 μm. Values are mean ± SEM. ***p < 0.0001 (t test). n = 30.