Figure 1.
ATE1 can directly target tubulin in vitro and is required for maintaining normal intracellular microtubule growth rate and stability. (A) Time course in vitro arginylation reaction showing Coomassie blue (top) and autoradiography (bottom). (B) In vitro arginylation on soluble tubulin and polymerized microtubules showing Coomassie blue (top) and autoradiography (bottom). (C) mCherry-EB3 signal (top) and EB3 tracks color-coded by mean straight-line speed ranging from 0 to 0.4 μm/s (bottom) from movies of Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells overexpressing mCherry-EB3 (Video 1). Scale bar = 20 μm. (D) Quantification of EB3 comet velocity. Mean ± SEM, N = 4 (n = 20 cells per condition), Ate1+/+ versus Ate1−/−: ***P = 0.0006, Ate1−/− versus Ate1−/−+ Ate1-GFP: ***P = 0.0003, one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. The violin plot represents the distribution of average EB3 comet velocity for individual cells. (E) Immunofluorescence images of Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells treated with 2 μM nocodazole for 0 or 10 min, extracted, and stained for ⍺-tubulin (magenta), GFP (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 = 365–567 cells per condition), 10 min Ate1+/+ versus Ate1−/−: **P = 0.002, 10 min Ate1−/− versus Ate1−/−+ Ate1-GFP: ***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 F1. Refer to the image caption for details.

ATE1 can directly target tubulin in vitro and is required for maintaining normal intracellular microtubule growth rate and stability. (A) Time course in vitro arginylation reaction showing Coomassie blue (top) and autoradiography (bottom). (B) In vitro arginylation on soluble tubulin and polymerized microtubules showing Coomassie blue (top) and autoradiography (bottom). (C) mCherry-EB3 signal (top) and EB3 tracks color-coded by mean straight-line speed ranging from 0 to 0.4 μm/s (bottom) from movies of Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells overexpressing mCherry-EB3 (Video 1). Scale bar = 20 μm. (D) Quantification of EB3 comet velocity. Mean ± SEM, N = 4 (n = 20 cells per condition), Ate1+/+ versus Ate1−/−: ***P = 0.0006, Ate1−/− versus Ate1−/−+ Ate1-GFP: ***P = 0.0003, one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. The violin plot represents the distribution of average EB3 comet velocity for individual cells. (E) Immunofluorescence images of Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells treated with 2 μM nocodazole for 0 or 10 min, extracted, and stained for ⍺-tubulin (magenta), GFP (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 = 365–567 cells per condition), 10 min Ate1+/+ versus Ate1−/−: **P = 0.002, 10 min Ate1−/− versus Ate1−/−+ Ate1-GFP: ***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 F1.

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