Fig. 6.

M1CY-positive bundles, but not M85CY-positive or M1Δ-positive bundles, are resistant to cold-shock or NaCl treatments. (A) HeLa cells expressing mutated spastin were subjected to cold shock for 30 min or incubated with 0.25 M NaCl for 30 min at 37°C and fixed. Scale bars: 20 µm. (B,C) HeLa cells expressing M1CY or M85CY were treated as described above and fixed after treatments (0 min), or at different time points (30, 60, 90, 120 and 180 min) after the recovery of temperature at 37°C (B) or the restoration of extracellular normal ionic strength (C). Untreated cells were taken as the control condition (Ctrl). HeLa cells were then stained for acetylated tubulin (see also Figs S5 and S7), and the average intensity of staining was measured in spastin-positive or nontransfected cells (nonpositive) and plotted over time. Values were normalized to untransfected cells before treatments. The number of cells analyzed ranged between 20 and 115. Data are shown as mean±s.e.m. Significance differences between M1CY and M85CY curves were determined by two-way ANOVA, Dunnett's post test. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001. (D,E) Representative immunoblot of HEK cells expressing GFP-tagged spastins or GFP as a control (data not shown) and treated by cold shock (D) or incubated with 0.25 M NaCl (E), as described above for HeLa cells. The lower panels show the quantification of acetylated tubulin/GAPDH ratio based on the integrated fluorescence intensity of the respective western blot bands and normalized to the values measured in GFP-expressing cells without treatment. Data represent the average of two independent experiments (mean±s.e.m.). Acetylated tub., acetylated tubulin.