Figure 7.
Depletion of Ate1 results in increased MAP1S association with spindle microtubules and increased mitotic index. (A–D) Immunofluorescence images of mitotic Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells stained for MAP1S (green), ⍺-tubulin (magenta), and DAPI (blue). Scale bar = 2 μm. Images are sum stacks. For each phase of the cell cycle, tubulin and MAP1S are scaled to the same respective intensity as the wild-type image for ease of visual comparison of intensity. (E) Quantification of MAP1S/⍺-tubulin during prometaphase. Mean ± SEM, N = 3 (n = 77–101 cells per condition), Ate1+/+ versus Ate1−/−: *P = 0.0286, Ate1−/− versus Ate1−/−+ Ate1-GFP: *P = 0.0462. (F) Quantification of MAP1S/⍺-tubulin during metaphase. Mean ± SEM, N = 3 (n = 40–42 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0098, Ate1−/− versus Ate1−/−+ Ate1-GFP: *P = 0.0132. (G) Quantification of MAP1S/⍺-tubulin during anaphase. Mean ± SEM, N = 3 (n = 20–48 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0027, Ate1−/− versus Ate1−/−+ Ate1-GFP: **P = 0.0095. (H) Quantification of MAP1S/⍺-tubulin during telophase. Mean ± SEM, N = 3 (n = 40–96 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0014, Ate1−/− versus Ate1−/−+ Ate1-GFP: ***P = 0.0006. For E through H, the statistical test is a one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Truncated violin plots represent the distribution of MAP1S/⍺-tubulin for individual cells. (I) Growth curve for Ate1+/+ and Ate1−/− cells. Mean ± SEM, N = 3 (n = 4 technical repeats for each condition), Day 9 Ate1+/+ versus Ate1−/−: **P = 0.0014, Day 9 Ate1+/+ versus Ate1−/−: **P = 0.0014, Day 10 Ate1+/+ versus Ate1−/−: **P = 0.0014, one-way RM ANOVA corrected for multiple comparisons. (J) Doubling rate calculated from Day 1–5 of the growth curve shown in I. In mitotic index experiments, cells were seeded at 10,000 cells/cm2, which falls into this range. Mean ± SD, N = 3, two-tailed paired t test. (K) Quantification of mitotic index for Ate1+/+ and Ate1−/− cells. Mean ± SEM, N = 3, **P = 0.0019, two-tailed paired t test. Refer to the image caption for details.

Depletion of Ate1 results in increased MAP1S association with spindle microtubules and increased mitotic index. (A–D) Immunofluorescence images of mitotic Ate1+/+, Ate1−/−, and Ate1−/− + Ate1-GFP cells stained for MAP1S (green), ⍺-tubulin (magenta), and DAPI (blue). Scale bar = 2 μm. Images are sum stacks. For each phase of the cell cycle, tubulin and MAP1S are scaled to the same respective intensity as the wild-type image for ease of visual comparison of intensity. (E) Quantification of MAP1S/⍺-tubulin during prometaphase. Mean ± SEM, N = 3 (n = 77–101 cells per condition), Ate1+/+ versus Ate1−/−: *P = 0.0286, Ate1−/− versus Ate1−/−+ Ate1-GFP: *P = 0.0462. (F) Quantification of MAP1S/⍺-tubulin during metaphase. Mean ± SEM, N = 3 (n = 40–42 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0098, Ate1−/− versus Ate1−/−+ Ate1-GFP: *P = 0.0132. (G) Quantification of MAP1S/⍺-tubulin during anaphase. Mean ± SEM, N = 3 (n = 20–48 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0027, Ate1−/− versus Ate1−/−+ Ate1-GFP: **P = 0.0095. (H) Quantification of MAP1S/⍺-tubulin during telophase. Mean ± SEM, N = 3 (n = 40–96 cells per condition), Ate1+/+ versus Ate1−/−: **P = 0.0014, Ate1−/− versus Ate1−/−+ Ate1-GFP: ***P = 0.0006. For E through H, the statistical test is a one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Truncated violin plots represent the distribution of MAP1S/⍺-tubulin for individual cells. (I) Growth curve for Ate1+/+ and Ate1−/− cells. Mean ± SEM, N = 3 (n = 4 technical repeats for each condition), Day 9 Ate1+/+ versus Ate1−/−: **P = 0.0014, Day 9 Ate1+/+ versus Ate1−/−: **P = 0.0014, Day 10 Ate1+/+ versus Ate1−/−: **P = 0.0014, one-way RM ANOVA corrected for multiple comparisons. (J) Doubling rate calculated from Day 1–5 of the growth curve shown in I. In mitotic index experiments, cells were seeded at 10,000 cells/cm2, which falls into this range. Mean ± SD, N = 3, two-tailed paired t test. (K) Quantification of mitotic index for Ate1+/+ and Ate1−/− cells. Mean ± SEM, N = 3, **P = 0.0019, two-tailed paired t test.

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