Mean distributions of kinetochores and microtubules during early prometaphase. (A) Schematics of the alignment approach. Each cell is individually rotated to orient the chromosome ring and translated to fix the position of spindle axis. (B) Spatial distribution of kinetochores (CenpF) and microtubules (α-tubulin) within the 210-nm equatorial slice (three 70-nm voxels deep) of the spindle. Images are maximum (CenpF) or mean (α-tubulin) intensity projections of early prometaphase cells rotated to orient the chromosome ring orthogonally to the view axis and translated to superimpose centers of the chromosome rings (crosses). (C) Normalized radially averaged α-tubulin intensity distribution profile from the spindle axis (0) toward the cell periphery. The peak is near the ring of centromeres (∼1.5 µm from the spindle axis). (D) Distribution of α-tubulin intensity near kinetochores. Images are mean intensity projections of kinetochores rotated to fix direction toward the center of the spindle (vertically downward) and translated to superimpose centroids of CenpF (outer kinetochore). Normalized heatmap suggests the highest α-tubulin intensity immediately in front of the kinetochore toward the spindle center (arrows). (E) Normalized α-tubulin intensity distribution from the CenpF centroid (0) toward the spindle center. (F–I) As in A–D, but the cells were treated with 20 nM GSK923295 to inhibit CenpE.