Astral MTs are destabilized upon disruption of the CHC-GTSE1 interaction. (A) Immunofluorescence images illustrating astral length and abundance (α-tubulin and EB1 staining) under labeled conditions. (B) Scheme illustrating the determination of “astral” EB1 comets and quantification method used to measure astral MT length and abundance in 3D reconstruction of cells from A. (C and D) Mean astral MT length (C) and Astral comet number per cell (D) are presented from conditions in A (n ≥ 31 cells per condition over n = 3 experiments pooled for analysis and presented as box and whisker plots; P values from Wilcoxon test adjusted for multiple comparisons [false discovery rate]). (E) Immunofluorescence images of U2OS cells transfected with control (Ctrl) or CHC siRNA (114 h). (F and G) Astral MT length (F) and abundance (G) in cells from E are presented (n ≥ 42 cells per condition over n = 4 experiments pooled for analysis and presented as box and whisker plots; P value from ANOVA and Wilcoxon test, respectively). (H) Immunofluorescence images of mESCs (α-tubulin and EB1 staining) transfected with control, mGTSE1, CHC, or mGTSE1 + CHC siRNA. (I and J) Astral MT length (I) and abundance (J) in cells from H are presented. To account for extreme variation in spindle shape after mGTSE1/CHC depletion in mESCs, the β threshold (see B) was calculated for each spindle based on its geometry (see Materials and methods; n ≥ 24 cells over n = 2 experiments pooled for analysis and presented as box and whisker plots; P values from ANOVA followed by Tukey’s test). n.s., not significant. (K) Stills from live-cell imaging of GTSE1^5xLID mCherry-β-tubulin U2OS cells with DNA stain, transfected with GTSE1 siRNA. Top and side view from a 3D reconstruction are presented. Scale bar: 10 µm. Letters indicate misaligned chromosomes. Notice misaligned chromosomes remaining outside of inner spindle mass when viewed in 3D. Numeric data is shown in Table S1.