Banerjee B , Kestner CA , Stukenberg PT .

GM063045 NIGMS NIH HHS , R01 GM063045 NIGMS NIH HHS

	Figure 1. EB1 localizes Aurora B to centromeres to phosphorylate kinetochores. (A) HeLa cells depleted of EB1 were immunostained with antiphospho-KNL1(Ser60) (pKNL1) and Aurora B antibodies. Bar, 2 µm. (B) Quantification of immunostaining intensities of pKNL1 shown in A. *, P = 2.0 × 10−127. (C) Centromeric Aurora B levels measured in control and EB1-depleted cells (n > 300 centromeres). *, P = 1.2 × 10−105. (D) EB1 depletion reduces Bub1-mediated phosphorylation of histone H2A (pH2AT120). Bar, 1.6 µm. (E) EB1 depletion reduces Bub1 at kinetochores. Bar, 2.2 µm. (F–H) Quantification of pH2AT120 (F), phospho–histone H3Thr3 (pH3T3; G), and Bub1 (H) levels in control and EB1-depleted HeLa cells (see Fig. S1 B for pH3T3 staining examples). The height of the boxes represents the interquartile range (IQR). The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. ACA, anticentromere antigen; a.u., arbitrary unit.
	Figure 2. EB1 localizes Aurora B at the centromere in a microtubule-dependent manner. (A) Loss of centromeric Aurora B is rescued by expressing siRNA-resistant EB1 (EB1siRes) but not the EB1K89E mutant (EB1K89EsiRes). Bar, 2.2 µm. (B) Loss of KNL1(Ser60) phosphorylation was rescued by expressing siRNA-resistant EB1 but not the EB1K89E mutant. Bar, 2.3 µm. (C) Quantification of immunostaining Aurora B intensities in A. *, P = 3.78 × 10−69; **, P = 2.54 × 10−75; ***, P = 1.33 × 10−35. (D) Quantification of immunostaining phospho-KNL1(S60) intensities in B. *, P = 5.23 × 10−42; **, P = 1.34 × 10−42; ***, P = 1.02 × 10−39. (E) Western blot of HeLa lysates showing endogenous and GFP-tagged EB1 and KNL1 levels (in kilodaltons). (Additional rescue experiments are shown in Fig. S2, A–C.) The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. a.u., arbitrary unit.
	Figure 3. Relationship of the EB1/microtubules and the histone phosphorylation pathways in CPC localization. (A) HeLa cells treated with 0.33 and 3.3 µM nocodazole for 7 h were fixed and stained with tubulin and Aurora B antibodies. The inset is a projection of six z sections. White arrowheads in 3.3 µM nocodazole (Noc; tubulin images) point to centrosomes. Bars: (main images) 1.8 µm; (inset) 0.22 µm. Settings that allow visualization of spindle microtubules obscure the microtubule foci in 0.33 µM nocodazole, so we have not shown control cells treated with DMSO. (B) Box and whisker plot of centromeric Aurora B levels measured in HeLa cells after the indicated treatments. The lines within the boxes represent the medians. Mean inner centromeric Aurora B levels from the same experiment shown in Fig. S3 A. *, P = 0.01182. (C) HeLa cells were treated with 10 µM reversine and 1 µM 5-iodotubericidine (HI) separately or in combination (reversine [R] + HI) in the presence of MG132. (Reversine only is shown in Fig. S3 B, and HI only with the same control cells is shown in Fig. S3 C.) To determine whether microtubules can recruit Aurora B in the absence of phosphohistone marks, HeLa cells were treated with or without 3.3 µM nocodazole along with the reversine, HI, and MG132 (reversine + HI + nocodazole). Control cells were treated with MG132 only. Cells were fixed after 30 min and stained with anti-pH3T3, anti-tubulin, and anti–Aurora B antibodies. Bright-field image (reversine + HI + nocodazole treatment) to show that there is a cell that lacks detectable staining. Bar, 1.7 µm. (D) Centromeric Aurora B levels were measured at indicated treatment conditions. In this experiment, 1 µM reversine was used. *, P = 6.65 × 10−127. (E) Expression of CENPB-INCENP fusion protein in U2OS-TR cells rescued the reduction of Bub1, phospho-KNL1, and pH3T3 levels after EB1 depletion. A stable U2OS-TR line was either mock treated (control) or EB1 siRNA treated with or without CENPB-INCENP induction. Bar, 1.8 µm. (F) Quantification of Bub1 levels. *, P = 1.05 × 10−262. (G) Quantification of phospho-KNL1(Ser60) levels. *, P = 8.95 × 10−31. Error bars show standard deviations. The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. ACA, anticentromere antigen; a.u., arbitrary units; res, resistant.
	Figure 4. EB1 is in close proximity to Aurora B at centromeres. (A) Microtubules stimulate Aurora B bound to INCENP790–856 (AI790–856) activity on MBP in vitro. The assay was performed with or without 3 µM taxol-stabilized microtubules (MT). (B) EB1 does not stimulate Aurora B kinase activity alone or in combination with microtubules. AI790–-856 in vitro kinase assay, using MBP as a substrate, showing the effect of adding 3 µM taxol-stabilized microtubules and 100 ng EB1 separately or in combination. Kinase activity of 20 nM AI790–856 was assayed similarly as in A. (C) Direct interaction between EB1 and the catalytic subunit of the CPC. Recombinant Xenopus GST–Aurora B bound to a fragment of INCENP790–856 (GST-AI) on glutathione–Sepharose 4 beads was incubated with the indicated concentration of recombinant xEB1, washed, and eluted with glutathione, and the two peak fractions of the elutions (E1 and E2) were quantified by immunoblotting. GST beads were used as a control. Molecular markers are given in kilodaltons. (D) Immunostaining of EB1 and Borealin in HeLa cells. (The single channel split of images is shown in Fig. S4 A.) Bar, 2.6 µm. (E) HeLa cells immunostained for Borealin and tubulin and the close proximity of EB1 and Aurora B shown by PLA. Insets illustrate PLA at individual centromeres. Prometaphase inset is a projection of four z sections, and metaphase inset is a projection of seven z sections (PLA controls are shown in Fig. S4, C and C′). Bars: (main images) 2.4 µm; (prometaphase inset) 0.39 µm; (metaphase inset) 0.35 µm. (F) Quantification showing percentage of occurrence of PLA spots proximal to centromeres from the PLA experiment shown in C. Error bars show standard deviations.
	Figure 5. Aurora B interacts with a distinct class of spindle microtubules. (A) Xenopus S3 cells immunostained for INCENP and tubulin (Tub) and the close proximity of tubulin and Aurora B (AurB) are shown by PLA. Insets are single z sections of Aurora B–tubulin interactions at the centromere (yellow boxes) and at the kinetochores (orange boxes). The bottom shows the effect of ice treatment on S3 cells before fixation. Bars: (main images) 11 µm; (insets) 0.51 µm. (B) Monastrol-treated Xenopus S3 cells immunostained for Ndc80 and tubulin and the close proximity of tubulin and Aurora B shown by PLA—projection of four z sections. Bars: (main images) 1.6 μm; (insets) 1 µm. (C) Nocodazole washout experiments were performed to show Aurora B specifically enriched on pre–K-fibers. Xenopus S3 cells were fixed and stained 5 min (projection of seven z sections) and 15 min (projection of nine z sections) after nocodazole washout. Bars: (main images) 2.3 μm; (insets) 1.4 µm. B and C insets are zoomed in views of the areas highlighted by the boxes showing Aurora B–tubulin PLA spots on pre–K-fibers. Aurora B–tubulin PLA signals are shown with Ndc80 and tubulin costaining. (Individual z slices of whole cell projections are shown in Videos 1–4.)
	Figure 6. EB1 and microtubules are required for Aurora B to phosphorylate kinetochores and chromatin substrates in prometaphase. (A) Experimental outline of B and C. (B) HeLa cells treated with ZM, MG132, and monastrol were washed out of ZM to monitor recovery of phospho–histone H3Ser10 (pH3S10) after 15 min. EB1-depleted cells are compared with control cells treated similarly and fixed immediately (ZM washout, 0 min) or replaced in ZM-free media and fixed after 15 min (ZM washout, 15 min). Bar, 1.6 µm. (C) Spreading of Aurora activity from centromeres to kinetochores (pKNL1) requires EB1. Note that centrosomal staining is an artifact, and kinetochore staining represents phospho-KNL1. Bar, 1.7 µm. (D) Quantification of the mean total pH3S10 recovery in B. *, P = 2.2 × 10−5. Error bars show standard deviations. (E) Quantification of kinetochore phosphorylation in C. (F) Centromeric Aurora B levels from B. **, P = 5.3 × 10−157. (G) Microtubules are required for the recovery of inner centromeric Aurora B and KNL1 phosphorylation after ZM washout. Note that the phospho-KNL1 has nonspecific staining of centrosomes (Welburn et al., 2010). Bar, 1.4 μm. (H) Quantification of inner centromeric Aurora B intensities in G (n > 160). Recov, recover; Noc, nocodazole. *, P = 4.19 × 10−91. (Scheme for the experiment is shown in Fig. S5 B.) The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. a.u., arbitrary units.
	Figure 7. Aurora B activity on chromatin is regulated by microtubules in prometaphase but not in prophase. (A) Phospho–histone H3Ser10 (pH3S10) and tubulin immunostaining of prophase and prometaphase cells at the indicated conditions. Insets show chromatin staining. AurB, Aurora B; w/o, washout. (Assay scheme is shown in Fig. S5 B.) Bars, 10 µm. (B) HeLa cells in prometaphase immunostained for tubulin, phospho–histone H3Ser10 (pH3S10), and chromatin at the indicated conditions. Bar, 2.2 µm. (C) Relative pH3S10 intensity plotted as a function of time after ZM washout in the presence or absence of nocodazole. Error bars show standard deviations. (D) Correlation between total cellular pH3S10 intensity from all time points and total tubulin intensity measured per cell (R2 = 0.91). Cellular pH3S10 intensities at 0 min and recovery phases in the absence of nocodazole (Noc) are represented as blue diamonds, and intensities from cells in nocodazole are shown in red squares (n = 58; data shown are one representative experiment of three repeats). MT, microtubule; a.u., arbitrary units.
	Figure 8. CPC activity spreads from centromeres to chromosome arms after nuclear envelope breakdown. (A) Assay scheme. (B) Cells that have endogenous survivin replaced with the survivinH80A mutant are unable to phosphorylate histone H3Ser10 in the ZM washout assay. HeLa cells stably expressing vector only, survivin-myc (siRNA resistant [res]), and survivinH80A-myc (siRNA resistant) were treated with survivin siRNA for 48 h followed by 1 h in ZM, MG132, and monastrol. Bar, 2.7 µm. (C) Mean total phospho–histone H3Ser10 intensity at 0 and 15 min in mock-treated (vector [vec.] only) and survivin siRNA-treated vector only (vector only + siRNA) and survivin-myc (wild type [WT] resistant)– and survivinH80A-myc (H80A resistant)–expressing cells. Error bars show standard deviations. a.u., arbitrary units. (D) Western blot showing Aurora B, survivin-myc, and endogenous survivin levels. Molecular mass markers are given in kilodaltons.
	Figure 9. Cooperation of histone phosphorylation and microtubules in centromeric localization of Aurora B. Model depicting the role of EB1 and microtubules (MT) in regulating inner centromeric Aurora B and its activity. Arrows do not imply direct interaction.

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