Kinetochores expand by forming micrometer-scale fibrous structures that extend throughout the chromosome mass in the absence of microtubules. (A–D) Comparison of mitotic chromosomes and kinetochores in human RPE-hTERT cells (A and B) and Xenopus egg extracts (C and D) in the presence (right) or absence (left) of 33 µM nocodazole to completely depolymerize microtubules (not depicted). Each image represents a set of chromosomes derived from a single nucleus. Centromeres are stained (green) with CREST antisera (A and B) or CENP-A (C and D) and costained for the outer kinetochore protein BubR1 (magenta) and DAPI (blue). All chromosomes (A and C; bars, 1 µm) and blowups of sister centromeres (B and D; bars, 0.2 µm) are shown at the same magnification to allow size comparison. (B and D) Colored arrows indicate lengths of maximum BubR1 signal perpendicular to the centromere–centromere axis (magenta) and the distance between centroids of centromere signals (green). White arrows indicate length measurements of traces along the filamentous structure beginning from the centroid of the nearest CENP-A focus. (E) Quantification of the total kinetochore staining on the chromosome cluster. Mean and standard deviation plotted in black. Mean values and fold change indicated above. Mann–Whitney test: ns, P > 0.5; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4–10 nuclei). (F) Staining of KMN network components (green) in nocodazole-treated extracts costained with BubR1 (magenta, chromosomes not depicted). Bars, 1 µm. All images are maximum intensity projections of 3D-SIM datasets. A.U., arbitrary unit.