Expressing R-β-actin in Ate1 ^−/− cells does not rescue microtubule growth rate or stability. (A) Schematic of expressing R-actin-GFP. Ub-R-β-actin-GFP is expressed, and the ubiquitin is immediately cleaved, producing R-β-actin-GFP. (B) Western blot showing the expression of R-β-actin-GFP in Ate1^−/− cells. (C) Images of Ate1^−/− cells co-transfected with mch-EB3 (top) and R-actin-GFP (bottom). Scale bar = 10 μm. (D) 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 Ate1^+/+ or Ate1^−/− cells overexpressing R-actin-GFP and/or mCherry-EB3 (Video 2). Scale bar = 20 μm. (E) Quantification of the EB3 comet velocity. Mean ± SEM, N = 3 (n = 9 cells per condition), Ate1^+/+ versus Ate1^−/−: ***P = 0.0031, Ate1^−/− versus Ate1^−/− + R-actin-GFP: ***P = 0.0033, 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. (F) Immunofluorescence images of Ate1^+/+, Ate1^−/−, Ate1^−/− + R-actin-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. (G) Quantification of the microtubule area per cell before nocodazole treatment. Mean ± SEM, N = 3 (n = 324–432 cells per condition), one-way RM ANOVA corrected for multiple comparisons. (H) Quantification of the average microtubule-positive area per cell normalized to t = 0. Mean ± SEM, N = 3 (n = 227–432 cells per condition), 10 min Ate1^+/+ versus Ate1^−/−: ***P = 0.0003, 10 min Ate1^−/− versus Ate1^−/− + Ate1-GFP: **P = 0.0021, two-way RM ANOVA corrected for multiple comparisons. For G and H, symbol colors indicate paired average measurements, and truncated violin plots represent the distribution of average microtubule-positive area for individual cells. Source data are available for this figure: SourceData FS4.