Fig. 3

SOD1 mutants impair polymerization of Golgi-derived microtubules. a. Images show NSC-34 cells expressing RFP or SOD1 variants tagged with RFP after extraction for soluble proteins and labeling with MannII-GFP (Golgi) and antibodies against α-tubulin (microtubules). Merged RFP/MannII-GFP images show that both wildtype SOD1 (arrow) and mutant SOD1 (arrowheads) localize to Golgi membranes. This is confirmed by Pearson’s correlation analysis (RFP-SOD1^wt 0.74 ± 0.09, RFP-SOD1^G85R 0.72 ± 0.09, RFP-SOD1^G93A 0.77 ± 0.14, RFP 0.13 ± 0.14, statistical significance SOD1 variants vs RFP : p < 0.0006 by student’s t-test). Mutant SOD1^G85R and SOD1^G93A specifically cause rarefaction of microtubules around fragmented Golgi profiles (arrowheads in lower panel). b. Biochemical fractionation of total (T), soluble (S) and polymerized (P) tubulins. Cells expressing SOD1^G85R or SOD1^G93A display a decreased ratio of polymerized detyrosinated (detyr-) tubulin. The ratio of polymerized α-tubulin is also decreased. Polymerization of β-actin is not affected by mutant SOD1. % P is equivalent to P/(P + S), * p < 0.01 by Mann–Whitney test, n = 3 experiments each. c. Flow cytometry of cellular microtubules in NSC-34 cells after extraction of soluble tubulins, microtubule stabilization and intracellular labeling with α-tubulin-FITC antibodies. Cells expressing SOD^wt (in grey, upper panel) display normal levels of α-tubulin-containing microtubules in comparison to cells expressing RFP (in black). Cells expressing mutant SOD1^G85R (in green, middle panel) or mutant SOD1^G93A (in blue, lower panel) show decreased levels of cellular microtubules. Median fluorescence signal per cell: 15.800 (RFP), 15.900 (SOD1wt), 11.600 (SOD1^G85R), 8.418 (SOD1^G93A). Statistical significance by chi square test, * T(x) > 200, ns T(x) = 0. d. Images showing transfected NSC-34 cells that were treated with Nocodazole (left column) and restored to drug-free medium for 12 min (right four columns). Cells were identified by RFP expression (not shown), Golgi profiles were identified by MannII-GFP and growing microtubules with antibodies against α-tubulin (pseudocolored in red). Mutant SOD1^G85R and SOD1^G93A impede regrowth of Golgi-derived microtubules (zoomed insets). Scale bar 5 μm. e. Diagrams showing reduced growth rate of Golgi-derived microtubules in cells expressing RFP- SOD1^G85R (upper panel) or RFP- SOD1^G93A (lower panel) as compared to cells expressing RFP- SOD1^wt or RFP. Microtubule length represents mean of mean of >12 cells per time point and condition and a total of 1495 microtubules analyzed. Statistical significance **** p < 0.0001 (RFP-SOD1^G85R vs RFP or vs SOD1^wt) and **** p < 0.0001 (RFP-SOD1^G93A vs RFP or vs SOD1^wt) by ANOVA test and Tukey’s multiple comparison test. f. Diagrams showing Taxol-mediated rescue of MannII-GFP-labeled Golgi fragmentation in cells expressing mutant SOD1^G85R or SOD1^G93A each tagged to RFP. Statistical significance * p < 0.01 (Taxol vs mock) by Mann–Whitney test, n ≥ 50 cells per well and 4 replicate wells were analyzed per condition