Polyglutamylation induces microtubule severing in HeLa cells. (A) MT morphology in HeLa cells expressing either active TTLL6 or an inactive mutant (TTLL6d). Transfected cells (green contours) were identified by CFP, which was coexpressed from the same plasmid (Fig. S2). Cells were stained with the general tubulin antibody 12G10, which detects α-tubulin in a glutamylation-independent manner (not depicted). Gray contours indicate examples of untransfected cells. Bar, 20 µm. (B) Quantification of MT mass in cells expressing active and inactive TTLL4, 6, and 11. Mean 12G10 fluorescence intensity was measured as indicated by contours in A for >30 cells per experimental condition and plotted as normalized to the mean of nontransfected cells (100%). Error bars indicate SD. Statistical significance was determined by two-tailed Student’s t test. All P values (**) are below 10−10. (C) Quantification as in B after expression of TTLL11 in cells transfected with nonsilencing control (scramble) or spastin-specific siRNA 1720 (transfection scheme of siRNA; see Materials and methods). (D) Validation of spastin siRNA. HeLa cells were transfected with scramble and spastin siRNA, and actin and spastin levels were detected on the same blot with specific antibodies. The siRNA 1720 reduces the levels of spastin to 13.2%.
