Skip to main content
. 2018 Nov 5;28(21):3408–3421.e8. doi: 10.1016/j.cub.2018.08.056

Figure 1.

Figure 1

Kinetochore Expansion Requires the RZZ Complex and SPDL1 but Is Independent of Dynein-Dynactin

(A) Cartoon showing the crescent shape characteristic of the expanded outer kinetochore, which encircles the compact inner kinetochore. Components analyzed in this figure are listed on the right.

(B) Immunofluorescence images showing that kinetochore expansion in nocodazole is inhibited after RNAi-mediated depletion of ROD or SPDL1. Scale bars, 5 μm; blow-ups, 1 μm.

(C–F) Quantification of relative kinetochore volume for CENP-E (C) and CENP-F (E) and kinetochore signal intensity for CENP-E (D) and CENP-F (F), based on fluorescence measurements in images as shown in (B). For each condition, the mean value per kinetochore was determined for individual cells. Final values are shown as the mean of mean (n = 20 cells), normalized to the control. Error bars denote the 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗∗p < 0.0001; ∗∗p < 0.01; ∗p < 0.05; not significant (ns), p > 0.05.

(G) Immunofluorescence images showing that depletion of the dynactin subunit DCTN1 does not affect kinetochore expansion. Scale bars, 5 μm; blow-ups, 1 μm.

(H and I) Quantification of relative kinetochore volume (H) and signal intensity (I), determined and plotted as described for (C)–(F). The t test was used to determine statistical significance. ∗∗∗∗p < 0.0001; ns, p > 0.05.

(J) Cartoon model of ZWILCH (PDB: 3IF8) obtained with PyMOL. Dotted lines mark loop regions that were not visible in the crystal structure and were added to improve clarity. The red boxed region indicates the position of E422 and D426.

(K) Immunofluorescence images showing that the ZWILCH mutant E422A/D426A does not support kinetochore expansion. Scale bars, 5 μm; blow-ups, 1 μm.

(L and M) Quantification of relative kinetochore volume (L) and signal intensity (M), determined and plotted as described for (C)–(F). The t test was used to determine statistical significance. ∗∗∗∗p < 0.0001.

See also Figure S1.