Wynne DJ , Funabiki H .

S10RR031855 NCRR NIH HHS , R01GM075249 NIGMS NIH HHS , S10 RR031855 NCRR NIH HHS , F32 GM103147 NIGMS NIH HHS , F32GM103147 NIGMS NIH HHS , R01 GM075249 NIGMS NIH HHS

	Figure 1. 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.
	Figure 2. CENP-C is a component of the expanded kinetochore. (A) Staining of CCAN proteins (green) in nocodazole-treated extracts costained with BubR1 (magenta, chromosomes not depicted). (B) Quantification of the total kinetochore staining on the chromosome cluster. Mean, standard deviation, and fold change indicated in black (n = 8–10 nuclei). (C) Western blots of chromosomes purified from untreated or nocodazole-treated extracts. Percent values calculated by correcting raw values with the ratio of H3 signal. (D) Schematic of Xenopus CENP-C protein and constructs used in D–I. (E) Mitotic chromosomes assembled in extracts in which the indicated 6×Myc–CENP-C construct was expressed. (F and G) Quantification of data from D, comparing the Myc signal to CENP-A or BubR1 staining. (H–J) Sample preparation and quantification as in E–G. Mann–Whitney test: ns, P > 0.5; *, P < 0.05; **, P < 0.01; ***, P < 0.001. All images are maximum intensity projections of 3D-SIM datasets; bars, 1 µm. A.U., arbitrary unit.
	Figure 3. The kinetics of expansion. (A) Examples of kinetochore staining at time points shortly after nuclear envelope breakdown (∼10 min after addition of fresh CSF extract to induce M phase entry). (left) Images of two nuclei at each time point to illustrate the types of figures seen at that time point. Bars, 1 µm. (right) Higher magnification images of a group of kinetochores from each nucleus. Note that after 8 min, small amounts of BubR1 have loaded only in nuclei that have lost their circularity and have presumably completed nuclear envelope breakdown (NEBD). In contrast, after 12 min, much more BubR1 staining can be seen, particularly in kinetochores in locations where the spindle is more disorganized (inset 3 and 4; bars, 0.2 µm). All images are maximum intensity projections of 3D-SIM datasets. (B and C) Quantification of individual kinetochore signals for the indicated protein over a time course during spindle assembly. Significance relative to the 45-min value. Mann–Whitney test: ***, P < 0.001; **, P < 0.01. n > 100 kinetochores from 5–8 nuclei. (D and E) Quantification of total signal from chromosome clusters for the indicated kinetochore component over a time course to indicate the rate of kinetochore expansion. Mean and standard deviation are plotted in black. A.U., arbitrary unit.
	Figure 4. Formation of the expanded kinetochore requires outer kinetochore proteins and CENP-C. (A) Model of kinetochore expansion. Proteins present on the expanded kinetochore are shown in magenta, whereas the core kinetochore proteins are in green. Question marks indicate sites of potential oligomerization. (B–G) Mitotic chromosomes assembled in Xenopus egg extracts after depletion indicated, treated with nocodazole, and stained for the kinetochore proteins indicated. Bars, 1 µm. (C, E, and G) Quantification of total signal from chromosome clusters in extracts from B, D, and F. P-values from Mann–Whitney test and percentage of mean value relative to ΔIgG are indicated above each condition. Mean and standard deviation are plotted in black. All images are maximum intensity projections of 3D-SIM datasets.
	Figure 5. CENP-C–dependent kinetochore expansion supports the SAC signal. (A) Extracts were immunodepleted of CENP-C and mixed with control extract to produce intermediate levels of CENP-C. (B) Mitotic chromosomes assembled in CENP-C titration extracts shown in A and treated with nocodazole. Bars, 1 µm. (C) Quantification of total signal for samples as in B. (D) Extracts described in A were assayed for checkpoint activity by challenging M-phase extract to cycle into interphase upon addition of calcium, which does not occur over 60 min in control extracts but occurred after 30 min in extracts with 10% and more rapidly in extracts with 0% of endogenous CENP-C. Cell cycle state was monitored by the presence of phosphorylated Threonine 3 of Histone H3 (H3T3ph). (E and F) Mitotic chromosomes assembled in nocodazole-treated control (ΔIgG) or CENP-C–depleted (ΔCENP-C) extracts and stained for the indicated checkpoint protein. (G and H) Quantification of total signal from E and F. Mean and standard deviation in black. Mann–Whitney test: ***, P < 0.0005; **, P < 0.01; *, P < 0.05. All images are maximum intensity projections of 3D-SIM datasets. AU, arbitrary unit.
	Figure 6. Aurora B is required for the core outer kinetochore, whereas Bub1 is specifically required for the expandable module. (A) Mitotic chromosomes assembled in Xenopus egg extracts immunodepleted of Bub1 kinase (ΔBub1) or the CPC using antibodies recognizing INCENP (ΔINC) and treated with nocodazole. (B) Quantification of the total signal, P values from Mann–Whitney test, and percentage of control mean value are shown. A.U., arbitrary unit. (C) Western blots of total extract (left) and chromosomes purified from nocodazole-treated control (ΔIgG) or CPC-depleted (ΔINC) extracts (right) show a reduction in CENP-C and a more severe loss of Ndc80, Mis12, and Zwint signal. (D) Mitotic chromosomes assembled in nocodazole-treated extracts. (left) Maximum intensity projections of whole nuclei. (right) Single optical section from the same dataset to highlight the inner centromeric staining underlying the expanded kinetochores (yellow arrows). (E) Mitotic chromosomes assembled in indicated extracts. Higher magnification images of a single optical section within the chromosome mass of Dasra-stained samples (area of inset shown with white boxes) are shown in the right-most panels to highlight the mislocalization of the CPC in all Bub1-depleted nuclei (cyan arrows) relative to controls (yellow arrows). wt, wild type. (F) Quantification normalized to the intensity of the BubR1 staining in control depletion (ΔIgG). Mean and standard deviation plotted in black. Mann–Whitney test: ***, P < 0.0001; ns, P = 0.28. All images are maximum intensity projections, except when “single section” is indicated, of 3D-SIM datasets. Bars: (A, D, and E, left) 1 µm; (E, right) 0.2 µm.
	Figure 7. A balance of kinase and phosphatase activity tunes the extent of expansion. (A–D) Mitotic chromosomes assembled in the indicated nocodazole-treated extract. (E) Mitotic spindles assembled in extracts treated with 1–3 µM PP1 inhibitor I-2 and processed for immunofluorescence against BubR1 (top, white; bottom, magenta). (F) Quantification of BubR1 signal for individual kinetochores, mean, standard deviation, and fold change in black. Mann–Whitney test: ***, P < 0.0001. All images are maximum intensity projections of 3D-SIM datasets; bars, 1 µm. A.U., arbitrary unit.
	Figure 8. Assembly of distinct functional modules within the kinetochore. (A) Comparison of the conventional view of kinetochore structure and assembly with our model of the kinetochore as distinct functional domains: an expandable and core module, each comprised of both inner and outer kinetochore components. Arrows indicate the direction of kinetochore assembly. (B) Schematic of the functional transition promoted by the dynamics of the expandable module.

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