Figure 6. Map1s depletion in cells... | Rockefeller University Press

Figure 6.

$Map1s depletion in cells overexpressing of Flag-TUBA1BE77Ais sufficient to rescue microtubule growth rate and stability. (A) Western blot for MAP1S (top) and tubulin (bottom) in soluble and polymerized tubulin fractions from wild-type cells overexpressing Flag-TUBA1BWT, Flag-TUBA1BE77A, TUBB5WT-Flag, or TUBB5D74A-Flag. Note that the tubulin loading control images in both panels (bottom) are the same as the ones in the supplemental panel S5E, since the two blots shown come from the same experimental repeat. (B) Quantification of the fraction of MAP1S associated with the polymerized tubulin. Mean ± SD, N = 3, Flag-TUBA1BWT versus Flag-TUBA1BE77A: *P = 0.042, two-tailed paired t test. Symbol colors indicate paired measurements. (C) mCherry-EB3 signal (top) and tracks color-coded by mean straight line speed ranging from 0 to 0.4 μm/s (bottom) from movies of wild-type cells overexpressing Flag-TUBA1BWT or Flag-TUBA1BE77A, siCtrl or siMap1s, and mCherry-EB3 (Video 6). Scale bar = 20 μm. (D) Quantification of the average EB3 comet velocity. Mean ± SEM, N = 3 (n = 15 cells per condition), Flag-TUBA1BWT + siCtrl versus Flag-TUBA1BE77A + siCtrl: **P = 0.004, Flag-TUBA1BE77A + siCtrl versus. Flag-TUBA1BE77A + siMap1s: **P = 0.002, one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Violin plot represents the distribution of average EB3 comet velocity for individual cells. (E) Immunofluorescence images of wild-type cells overexpressing Flag-TUBA1BWT or Flag-TUBA1BE77A and siCtrl or siMap1s and treated with 0.5 μM nocodazole for 0 or 10 min, extracted, fixed in methanol, and stained for Flag (magenta), ⍺-tubulin (green), and DAPI (blue). Scale bar = 10 μm. (F) Quantification of the average microtubule-positive area per cell normalized to t = 0. Mean ± SEM, N = 3 (n = 112–194 cells per condition), 10 min Flag-TUBA1BWT + siCtrl versus Flag-TUBA1BE77A + siCtrl: ***P = 0.001, 10 min Flag-TUBA1BE77A + siCtrl versus. Flag-TUBA1BE77A + siMap1s: ***P = 0.001, two-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. The truncated violin plot represents the distribution of the average microtubule-positive area for individual cells. Source data are available for this figure: SourceData F6. Refer to the image caption for details.$

Map1s depletion in cells overexpressing of Flag-TUBA1B ^E77A is sufficient to rescue microtubule growth rate and stability. (A) Western blot for MAP1S (top) and tubulin (bottom) in soluble and polymerized tubulin fractions from wild-type cells overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag. Note that the tubulin loading control images in both panels (bottom) are the same as the ones in the supplemental panel S5E, since the two blots shown come from the same experimental repeat. (B) Quantification of the fraction of MAP1S associated with the polymerized tubulin. Mean ± SD, N = 3, Flag-TUBA1B^WT versus Flag-TUBA1B^E77A: *P = 0.042, two-tailed paired t test. Symbol colors indicate paired measurements. (C) mCherry-EB3 signal (top) and tracks color-coded by mean straight line speed ranging from 0 to 0.4 μm/s (bottom) from movies of wild-type cells overexpressing Flag-TUBA1B^WT or Flag-TUBA1B^E77A, siCtrl or siMap1s, and mCherry-EB3 (Video 6). Scale bar = 20 μm. (D) Quantification of the average EB3 comet velocity. Mean ± SEM, N = 3 (n = 15 cells per condition), Flag-TUBA1B^WT + siCtrl versus Flag-TUBA1B^E77A + siCtrl: **P = 0.004, Flag-TUBA1B^E77A + siCtrl versus. Flag-TUBA1B^E77A + siMap1s: **P = 0.002, one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Violin plot represents the distribution of average EB3 comet velocity for individual cells. (E) Immunofluorescence images of wild-type cells overexpressing Flag-TUBA1B^WT or Flag-TUBA1B^E77A and siCtrl or siMap1s and treated with 0.5 μM nocodazole for 0 or 10 min, extracted, fixed in methanol, and stained for Flag (magenta), ⍺-tubulin (green), and DAPI (blue). Scale bar = 10 μm. (F) Quantification of the average microtubule-positive area per cell normalized to t = 0. Mean ± SEM, N = 3 (n = 112–194 cells per condition), 10 min Flag-TUBA1B^WT + siCtrl versus Flag-TUBA1B^E77A + siCtrl: ***P = 0.001, 10 min Flag-TUBA1B^E77A + siCtrl versus. Flag-TUBA1B^E77A + siMap1s: ***P = 0.001, two-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. The truncated violin plot represents the distribution of the average microtubule-positive area for individual cells. Source data are available for this figure: SourceData F6.