Figure 9.

High levels of C448Y spastin overexpression decrease MT repolymerization. High levels of mutated spastin isoforms were expressed in RFL-6/Tet-On cells transiently transfected with pTRE/M87CY or pTRE/M1CY and cultured for 18 h with of 0.5 μg/ml Dox. After 15 min of depolymerization in the presence of 2 μg/ml nocodazole, cells were rinsed and cultured for 20 min in nocodazole-free medium to allow MT repolymerization. Staining with anti-spastin antibody identified spastin-expressing cells. Tubulin staining was used to evaluate MT levels. A, High levels M87 C448Y (cell outlined in red) moderately decreased MT repolymerization compared with nonexpressing cells (outlined in yellow). B, In cells expressing aggregated M1 C448Y (outlined in red), decrease of MT repolymerization correlates directly with the levels of M1 C448Y expression. C, High levels of M1 C448Y decorating MTs (in cell outlined in red) can also decrease repolymerization. Scale bar, 20 μm.