The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere.
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We thank J. Ellenberg, C. Brangwynne, G. Narlikar and D. Foltz for reagents, and D. Burke and S. Mattada for discussions. P.T.S. and P.T. were funded by NIH grant numbers R01GM124042 and R24OD023697. F.P. and M.A.D. were supported in part by a grant from the NSF (MCB-1615701). M.A.D. and E.G. were funded by NIH grant number P41-GM103540.
The authors declare no competing interests.
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Integrated supplementary information
(a) Superdex-200 gel filtration profile of ISBWT (red), ISBΔ139-160 (green), and ISBΔ165-180 (blue) broken grey line shows the profile of the FPLC standard. Brown bar indicated the fractions from gel filtration run on SDS-PAGE and stained with Coomassie brilliant blue (b). For a and b, the experiment was performed once for each protein prep. Source data for a are shown in Supplementary Table 2.
(a) DIC micrographs of the Xenopus. laevis INCENP1-58-Survivin-Dasra (ISD) complex coacervates under indicated conditions. 3 independent repeats. (b) Circularity of the GFP-ISB coacervates from experiment shown in Fig. 1d. n=257 and n=369 coacervates for 50 mM NaCl and 5% PEG respectively. Red line represents mean of the data. (c) Surface wetting and shearing of the ISB coacervates (orange arrow) under the indicated conditions. (d) Micrographs showing ISB coacervates under increasing NaCl concentration. (e) Graph showing area of the coacervates from d under increasing NaCl concentration (n=60, 71, 98 and 91 coacervates were analyzed for 50, 75, 100 and 125 mM NaCl respectively). (f) Micrographs showing ISB coacervates under increasing ISB concentration. (g) Graph showing area of the coacervates from F under increasing concentration of the ISB (n=74, 89, 58 and 98 coacervates were analyzed for 5,11, 21 and 42 μM ISB respectively). (h) Micrographs of ISB coacervates formed in presence of increasing concentration of PEG-3350. (i) Graph showing area of coacervates under increasing concentration of PEG-3350 (n=105, 182 and 149 coacervates were analyzed for 2,3 and 5% PEG respectively). Combined data from 3 independent experiments was reported for e,g and i. n.s. P>0.05, *** P<0.0001. Box and whisker graphs: median (central line), 25th-75th percentile (box), and 5th-95th percentile (whiskers). For statistical analysis in e, g, and i Kruskal-Wallis test was applied followed by Dunn’s multiple comparison test. (j and k) Electron micrographs of ISB coacervates formed under indicated conditions showing highly circular coacervates and coacervates arrest mid-fusion due to gelation. A few apparently aggregated networks could also be observed under condition of low salt and are shown as contrast to spherical coacervates. (l) DIC micrographs of 20 μM ISB showing coacervates arrest mid-fusion (yellow arrow). (m) Control from GFP-ISB FRAP analysis (Fig. 1f and Fig. 2a, b) showing no significant bleaching of GFP-ISB coacervates over the duration of imaging. For c-m all experiments were repeated 3 times independently. Scale bar 5 μm except for j and k where it is 1 μm and for the middle panel in k it is 2 μm. Source data for b, e, g and i are shown in Supplementary Table 2.
Supplementary Figure 3 FCS measurements of GFP-Borealin in the cytoplasm and at the inner-centromere.
(a) Equations used to fit the FCS correlation curves. b) The residuals of FCS fitted curves for GFP-Borealin in the cytoplasm of mitotic cell and at the inner-centromere (c) of the mitosis. (d) Graph showing diffusion coefficients of the fast population of GFP-Borealin inside the cytoplasm (Grey) or inner-centromere (Blue) during mitosis (n=25 regions were analyzed in both inner-centromere and cytoplasm). n.s. indicates P= 0.37. (e) Graph of Gslow/Gfast for GFP-Borealin in the cytoplasm (Grey) or inner-centromere (Blue) of a mitotic cell (n=25 regions were analyzed in both inner-centromere and cytoplasm). *** indicates P= 2.38E-04. For d and e statistical analysis was performed by applying two-tailed unpaired Mann-Whitney test. (f) FCS Autocorrelation functions of Inner centromere populations DFast (black line), DSlow (red line) and FCS autocorrelation curve in coacervates (green line). For b-f combined data from three independent repeats. For the whole figure same data as shown in Fig. 2b was analyzed. Box and whisker graphs: median (central line), 25th-75th percentile (box), and 5th-95th percentile (whiskers). Source data for b-f are shown in Supplementary Table 2.
Supplementary Figure 4 Enrichment of macromolecules in ISB phase and nucleation of microtubules from the ISB coacervates.
Representative images and graph showing partition coefficient of (a) Histone H3.3 mono-nucleosome (n=98), (b) Cy3-azide (n=33) and (c) GFP-xMad2 (n=101) in ISB phase. (d) Control images for experiment show in Fig. 3a. (e) Representative images and graph showing partition coefficient of rhodamine-α/β-tubulin dimer (n=111). (f) Images showing nucleation of microtubules upon enrichment of Cy5-α/β-tubulin dimers in ISB coacervates. Nucleation was observed from ISB coacervates at 1.25 uM α/β-tubulin dimers concentration in presence of GTP and not seen in the absence of GTP, the experiment was repeated independently twice. Yellow arrow points to the microtubules. “n” is the number of coacervates analyzed per condition. Box and whisker graphs: median (central line), 25th-75th percentile (box), and 5th-95th percentile (whiskers). Scale bar 5 μm. Source data for a-c and e are shown in Supplementary Table 2.
Supplementary Figure 5 Effect of increased monovalent cation on stability of ISB coacervates and Cat-granule profiles of CPC components.
(a) Graph showing enrichment of mCherry signal in the foci at 95 s. Data from experiment shown in Fig. 5a (n=99 puncta (11 cells) for BorWT-mch-cry2 and n=42 puncta (11 cells) for Bor139-160-mch-cry2; 17 cells were analyzed for mch-cry2 alone; the experiment was repeated twice). Middle brown line indicates mean and error bars indicate s.d.. For statistical analysis two tailed unpaired T-test with Welch’s correction was used, *** P<0.0001. (b) Graph showing average DIC (top) and mCherry (bottom) intensities along the line across a lacO arrays from experiment shown in Fig. 5b (n=22 LacO spots for mCherry-LacI-Borealin and n=23 LacO spot for mCherry-LacI; the experiment was repeated twice). Error-bars are s.e.m., the black dotted line in DIC graph is a reference line showing a flat profile. (Top right) insets from Fig. 5b with cyan arrow showing an example of line and its direction along which the intensities were measured and plotted. (c) DIC micrographs under indicated conditions showing disruption of ISB coacervates by 3% 1,6-Hexanediol (same conditions as in Fig. 5c, d). (d) DIC micrographs under indicated conditions showing disruption of ISB coacervates by 90 mM NH4OAC (same conditions as in Fig. 5e, f). (e) DIC micrographs of ISB coacervates in buffer containing indicated amount of NaCl and PEG-3350 (same condition as in Fig. 5g, h). For c-e, the experiment was repeated twice. (f) Graph showing normalized fluorescent intensity of Aurora-B-GFP at the inner-centromeres under indicated conditions from experiment shown in Fig. 5e (n=318, 430, 412 and 892 inner-centromeres were analyzed for 25 mM NaCl, 220 mM NaCl, 25 mM NaCl + 10% PEG-3350, and 220 mM + 10% PEG-3350 respectively). For statistical analysis two-tailed T-test with Welch’s corrections was applied, ***P<0.0001. Box and whisker graphs: median (central line), 25th-75th percentile (box), and 5th-95th percentile (whiskers). Experiment in f was repeated twice independently. (g) Catgranule profile of Borealin, Survivin and INCENP showing high propensity of Borealin central domain to participate in phase-separation. Orange and brown shaded region shows 139-160 and 165-180 amino acids of Borealin respectively. (h) PONDR profile of Borealin showing disorder propensity of the protein, Orange and brown shaded region shows 139-160 and 165-180 amino acids of Borealin respectively. (i) Alignment of Borealin region predicted to drive granule formation. Orange and brown box indicate the putative regions diving granule formation. Red and blue arrows indicate residues mutated in Borealin8A and Borealin13A mutants respectively. Scale bar is 5 μm. Source data for a, b and f are shown in Supplementary Table 2.
Supplementary Figure 6 Phase-separation property of the CPC is important for its inner-centromere localization.
(a and b) Western blots showing endogenous Borealin, LAP-Borealin and Tubulin levels. Cells were treated as indicated in Fig. 6f, except for treatment with 3.3 μM Nocodazole. Lysates for western blotting were collected 8 hours after second thymidine release. (c) Western blot showing relative levels of endogenous Borealin to LAP-BorealinWT or LAP-BorealinΔ139-160, cells were treated with 3.3 μM Nocodazole and arrested for 20 hours to enrich for mitotic cells, followed by cell lysis and western blot. (d) Micrographs showing staining of INCENP and ACA in cells rescued with either LAP-BorealinWT or LAP-BorealinΔ139-160. Cells were treated as shown in Fig. 6f, with the exception of the nocodazole treatment. For a-d the experiment was performed once. (e) Graph showing normalized intensity (normalized to that in cells expressing LAP-BorealinWT) of INCENP/ACA from experiment shown in d. (n=172 and n=157 inner-centromeres for LAP-BorealinWT and LAP-BorealinΔ139-160 respectively). Micrographs showing staining of H3T3ph (f), H2ApT120 (h), hSgo1 (j) and ACA in cells rescued with either LAP-BorealinWT or LAP-BorealinΔ139-160; cells were treated as shown in Fig. 6f. For f,h and j experiment was repeated twice. Graph showing normalized intensity (normalized to that in cells expressing LAP-BorealinWT) of H3T3ph (n=210 inner-centromeres from 10 cells for LAP-BorealinWT, n=170 inner-centromeres from 8 cells for LAP-BorealinΔ139-160, P=0.0005) (g), H2AT120ph (n=214 centromeres from 10 cells for LAP-BorealinWT, n=172 centromeres from 10 cells for LAP-BorealinΔ139-160, P=0.3494), (i), and hSgo1 (n=173 centromeres from 7 cells for LAP-BorealinWT, n=153 centromeres from 8 cells for LAP-BorealinΔ139-160, P=0.3094), (k) from experiment shown in f,h,and j respectively. (l) Micrographs of ISBWT and ISBΔ139-160 coacervates formed under indicated condition showing enrichment of CF555-hSgo11-112. Experiment repeated twice. (m) Graph showing partition coefficient of CF555-hSgo11-112 in ISBWT (n=104 coacervates) and ISBΔ139-160 (n=66 coacervates) from experiment shown in l, combined data from two independent experiments, P=0.092. For statistical analysis in e,g,i and k two-tailed T-test was applied with Welch’s correction, n.s. P>0.05, *** P<0.0001. All box and whisker graphs represent the median (central line), 25th-75th percentile (bounds of the box), and 5th-95th percentile (whiskers). Scale bar is 5 μm. For a-e experiment was done once. For f-k experiment was repeated twice. Source data for e, g, i, k and m are shown in Supplementary Table 2. Unprocessed blots for a-c are shown in Supplementary Fig. 8.
Supplementary Figure 7 Phase-separation by the CPC is important for maintenance of taxol induced mitotic arrest but only marginally affects the time to align chromosomes.
Cumulative frequency plot of the duration of Nuclear envelop break down (NEBD) to metaphase (a), metaphase to anaphase (b), and NEBD to anaphase (c); data from experiment shown in Fig. 7a, b. For statistical analysis two-tailed T-test with Welch’s correction was applied. (d) Cumulative frequency plot of the duration of mitotic arrest in presence of 100 nM paclitaxel from Fig. 7d–f. For statistical analysis two-tailed Mann-Whitney test was applied. Mean ± SEM is shown in parenthesis and “n” indicating the number of cells analyzed. Source data for a-d are shown in Supplementary Table 2.
(a-c) Uncropped blots from the indicated figure. Dotted box indicates the cropped area shown in the figure.
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Trivedi, P., Palomba, F., Niedzialkowska, E. et al. The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nat Cell Biol 21, 1127–1137 (2019). https://doi.org/10.1038/s41556-019-0376-4
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