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	FIGURE 1:. Direct binding of the Borealin C-terminal dimerization domain to Sgo1. (A) Schematic of the chromosomal passenger complex (CPC), which includes Aurora B kinase (AURKB), INCENP, Borealin-2, and Survivin. Aurora B kinase binds to the C-terminal INbox domain of INCENP, and Borealin-2 and Survivin bind to the N-terminal centromere targeting domain of INCENP. Borealin-2 contains three domains: an N-terminal coiled-coil domain through which it interacts with Survivin and INCENP, an unstructured central loop region that binds to nucleosomes, and a C-terminal dimerization domain. Sgo1 contains three domains: an N-terminal coiled-coil domain, a middle domain, and a C-terminal basic domain that includes the Sgo1 motif that interacts with H2AT120ph nucleosomes. (B) Yeast two-hybrid (Y2H) interactions of Sgo1 fragments with Borealin, Borealin-2, and INCENP. White colonies on the left are representative of diploid growth (DDO); blue colonies on the right represent interactions occurring on selective media (DDOXA). Borealin and Borealin-2 interact with Sgo11–150 but not Sgo1151–663. INCENP shows no interaction with Sgo1. Images are representative of four separate experiments. Representative positive and negative Y2H controls used throughout are shown in Supplemental Figure S1B. (C) Y2H interactions of Borealin-2 fragments with Sgo11–150 and Survivin. Borealin-21–144 and Borealin-21–235 interact with Survivin but not Sgo11–150, even under low stringency, and Borealin-2216–296 interacts with Sgo11–150. Images are representative of four separate experiments. See also Supplemental Figure S1, C and D. (D) Binding assay showing GST-Borealin-2216–296 interaction with Sgo11–150 -His6-MBP. Colloidal Coomassie staining of SDS–PAGE gel including input and beads is shown. The input represents 7% total Sgo11–150-His6-MBP incubated with beads. (E) Equilibrium assay to measure affinity of GST-Borealin-2216–296 for Sgo11–150-His6-MBP. Increasing amounts of GST-Borealin-2216–296-bound glutathione sepharose 4B beads were titrated into Sgo11–150-His6-MBP at 2 μM. Beads were pelleted, and samples of the supernatant were separated by SDS–PAGE to determine the fraction bound (see Materials and Methods and Supplemental Figure S1E). Curve is best fit of data to one-site binding equation yielding equilibrium constant of 10 μM. (F) Protein sequence alignment of the dimerization domains from Borealin-2 and Borealin-1 from indicated species. Clustalx coloring was applied to indicate conservation. Numbering denotes amino acid positions from X. laevis Borealin-2. Red circles mark amino acids featured in Figure 1G and mutated in Figure 1H. (G) Homology model of the X. laevis Borealin-2 (CDCA9/Dasra A) dimerization domain based on PDB ID: 2KDD (Bourhis et al., 2009), showing surface-exposed acidic residues. Each monomer is indicated by separate colors. (H) Binding assay showing how mutations in Borealin-2 dimerization domain affect GST-Borealin-2216–296 interaction with Sgo11–150-His6-MBP. Coomassie staining of SDS–PAGE gel including input and beads is shown. The input represents 10% of total Sgo11–150-His6-MBP incubated with beads. (I) Binding assay showing GST-Borealin-2216–296 interaction with Sgo1-His6-MBP truncations. Colloidal Coomassie staining of SDS–PAGE gel including input and beads is shown. The input represents 10% Sgo1-His6-MBP variants incubated with beads.
	FIGURE 2:. Borealin and PP2A can bind simultaneously to the Sgo1 coiled coil. (A) Sequence alignment of the N-terminal coiled-coil region of Sgo1 in indicated species. Clustalx coloring was applied to indicate conservation in sequence alignment. Numbering denotes amino acid positions from X. laevis Sgo1. Red circles indicate amino acids mutated in Sgo11–150-3A, a mutant that cannot bind to PP2A. (B) Y2H interactions of Borealin-2 with Sgo11–150 and Sgo11–150-3A (Y57A, N60A, T62A). Borealin-2 interacts with both Sgo11–150 and Sgo11–150-3A. Images are representative of four separate experiments. See also Supplemental Figure S1F. (C) Y2H interactions of Sgo11–150-3A with Sgo11–150-3A. Sgo11–150-3A interacts with itself. Images are representative of four separate experiments. (D) Western blot for proteins that copurify with Sgo11–150-GFP or Sgo11–150-3A-GFP (Y57A, N60A, T62A) expressed from mRNA in Xenopus egg extracts. IP, immunoprecipitated. (E) Schematic of co-binding experiment performed to investigate whether Sgo151–150-His6-MBP can bind to GST-Borealin-2216–296 and PP2A complex simultaneously. (F) Western blot for proteins that co-purify with GST or GST-Borealin-2216–296 in co-binding experiment. Ponceau indicates loading of GST or GST-Borealin-2216–296.
	FIGURE 3:. The Borealin–Sgo1 interaction controls CPC loading onto chromosome arms to promote spindle assembly. (A) Schematic of Borealin-2 constructs used to express mRNAs in Xenopus egg extract, along with full-length versions of other CPC components, Aurora B, INCENP, and Survivin. All four constructs were expressed to reconstitute the CPC in ΔCPC extracts. (B) Western blot for INCENP, GFP, Aurora B, Borealin-2, Survivin, histone H3 phosphorylation (H3T3ph), and tubulin for samples from Xenopus wild-type and or CPC-depleted (ΔCPC) metaphase extracts with indicated CPC variants expressed from mRNA. (C) Representative immunofluorescence (IF) images of replicated chromosomes in wild-type and ΔCPC metaphase extracts with indicated CPC variants (Western blot for samples shown in B). Chromosomes were stained for INCENP and Borealin-2-GFP. See also Supplemental Figure S2A. (D) Quantification of fluorescence intensity of INCENP was normalized to wild-type condition. n = 50 chromatin structures per condition. Error bars represent SD unless otherwise noted, and asterisks indicate a statistically significant difference (*, p < 0.001). A.U., arbitrary units. (E) Quantification of fluorescence intensity of Borealin-2 GFP was normalized to Borealin-2-GFP condition. n = 50 chromatin structures per condition. (F) Top: Representative IF images of spindles formed in wild-type and ΔCPC extracts with indicated CPC conditions. Rhodamine-labeled tubulin was added to visualize microtubules (white). Bottom: Chromatin was stained with Hoechst (white), and rhodamine-labeled tubulin was added to visualize microtubules (red). (G) Quantification of bipolar spindle length. n = 50 spindles per condition.
	FIGURE 4:. Artificial dimerization of Borealin is sufficient for CPC-mediated BubR1 enrichment at kinetochores. (A) Representative IF images of replicated chromosomes in wild-type and ΔCPC metaphase extracts with indicated CPC variants (Western blot for samples shown in Figure 3B). Kinetochores were stained for BubR1. (B) Quantification of fluorescence intensity of BubR1 was normalized to wild-type condition. n = 100 kinetochores per condition.
	FIGURE 5:. The CPC is recruited to chromatin through multiple mechanisms. Model for the multivalent recruitment of the CPC to chromatin. Survivin interacts with the histone H3T3ph tail (Kelly et al., 2010; Du et al., 2012; Niedzialkowska et al., 2012; Jeyaprakash et al., 2011), the Borealin N-terminus and its central loop interact with nucleosomes (Abad et al., 2019), and the Borealin C-terminal dimerization domain interacts with Sgo1, which in turn interacts with histone H2AT120ph C-termini (Liu et al., 2015b; Kawashima et al., 2010) to recruit the CPC (the CEN module of the CPC is shown here: the N-terminus of INCENP, Survivin, and Borealin) to chromatin. It is currently unclear whether the H3T3ph and H2AT120ph marks are present on the same nucleosome. The N-terminal coiled-coil region of Sgo1 can interact with Borealin-2 and PP2A simultaneously, suggesting that both the CPC and PP2A may be recruited to the same nucleosome.

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