Huang Y , Li T , Ems-McClung SC , Walczak CE , Prigent C , Zhu X , Zhang X , Zheng Y .

R01 GM059618 NIGMS NIH HHS , R01 GM106023 NIGMS NIH HHS , R01 GM056312 NIGMS NIH HHS , R01 GM110151 NIGMS NIH HHS , R35 GM122482 NIGMS NIH HHS

             mitotic spindle kinetochore microtubule

	Figure 1. Effects of BuGZ on AurA kinase activity in mitosis. (A–D) Cells were transfected with control, BuGZ, or TPX2 siRNA (si) or were treated with AurA inhibitor MLN8237 (100 nM) followed by staining with antibodies to total AurA (A) or p-AurA (B). Bars, 10 µm. The immunostaining intensity of total AurA (C) and p-AurA (D) in control siRNA–treated cells in metaphase or prometaphase (ProM, containing misaligned chromosomes or thick chromosome bars) and cells in the experimental groups containing misaligned chromosomes were quantified. 75–152 total cells from three independent experiments in each experiment were measured and quantified. Error bars indicate SEM. One-way ANOVA: *, P < 0.05; ***, P < 0.001. (E) Scheme of cell synchronization and mitotic shakeoff. (F) Cells transfected with siRNA were synchronized, collected, and analyzed by Western blotting with indicated antibodies. (G) XEEs were depleted (dep) with unimmunized rabbit IgG (IgG), xBuGZ, or xTPX2 antibodies or were treated with MLN8237. xAurA was immunoprecipitated and analyzed by Western blotting using indicated antibodies. (H) XEEs were depleted of xBuGZ and supplemented by purified YFP or YFP-xBuGZ followed by AurA immunoprecipitation and Western blotting. Noc, nocodazole; Thy, thymidine.
	Figure 2. BuGZ contributes to MT assembly and reduces S196 phosphorylated xMCAK. (A) MT asters and spindles assemble around AurA beads at the indicated time points in mock-depleted XEEs or XEEs depleted (dep) of xBuGZ or xTPX2 or treated with MLN8237. Bar, 10 µm. (B and C) MT aster lengths (B) and percentages of bipolar spindles (C) were quantified. Approximately 50 (B) and ∼200 (C) AurA bead structures were quantified in three independent experiments. (D) XEE mock-depleted by unimmunized rabbit IgG, xBuGZ, or xTPX2 antibodies or treated with MLN8237 were analyzed by Western blotting probing with the indicated antibodies. The means and SEM are graphed. Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
	Figure 3. The zinc fingers of BuGZ directly bind to the kinase domain of AurA. (A and B) Immunoprecipitation (IP) using BuGZ (A) or AurA (B) antibodies in mitotic HeLa cell lysates. Equivalent of 1% of the input lysate and 20% of total immunoprecipitates were loaded. (C and D) Immunoprecipitation using xBuGZ (rabbit polyclonal IgG; r IgG; C) or xAurA (mouse polyclonal IgG; m IgG; D) antibodies in XEEs. Equivalent of 0.2% of the input lysate and 30% of total immunoprecipitates were loaded. (E) Schematic of xBuGZ with NLS, zinc fingers (ZnFs), and the C-terminal region as well as YFP-tagged xBuGZ used in the study. (F) Purified YFP, YFP-xBuGZ, YFP-xBuGZΔ7, YFP-xBuGZΔN58, or YFP-xBuGZΔN92 were incubated with purified xAurA, followed by pulldown with antibody to YFP and then Western blotting and Coomassie Brilliant blue (CBB) staining. (G) Purified GST or GST-xBuGZ(8–58) was incubated with purified xAurA in the presence or absence of EDTA followed by GST pulldown, Western blotting, and Coomassie blue staining. (H) Purified GST, GST-xAurA, GST-xAurA(1–25), GST-xAurA(26–126), or GST-xAurA(127–407) were incubated with YFP or YFP-xBuGZ followed by GST pulldown, Western blotting, and Coomassie blue staining. For G and H, 0.1% of total reaction and 10% of total xBuGZ pulldowns were analyzed by Western blotting. 1% of total reaction and 10% of total pulldowns were analyzed by Coomassie blue staining.
	Figure 4. Effects of BuGZ phase separation on AurA binding. (A and B) Purified YFP-xBuGZ or YFP-xBuGZΔN58 was incubated to allow coacervation in the presence of purified mCherry-xAurA (A) or mCherry (B). Bars, 10 µm. (C) Purified YFP, YFP-xBuGZ, or YFP-xBuGZ13S were incubated with purified GST or GST-xAurA at RT or 4°C followed by GST pulldown, Western blotting (0.1% of total reaction and 10% of total YFP-xBuGZ and YFP-xBuGZ13S pulldowns were analyzed), and Coomassie Brilliant blue (CBB) staining (1% of the total reaction and 10% of total pulldowns were analyzed). (D) Schematic of droplet spindown assay. (E–G) Quantification of YFP-xBuGZ and xAurA (E), YFP-xBuGZΔN58 and xAurA (F), and YFP-xBuGZ13S and xAurA (G) in the pellets (X axis) against the initial input xBuGZ proteins (Y axis). Error bars indicate SEM.
	Figure 5. Effects of BuGZ phase transition and binding to AurA on kinase activity. (A) Purified xAurA (cleaved from mCherry-xAurA) was incubated from purified xBuGZ (cleaved from YFP-xBuGZ) and MBP to assay AurA kinase activity in vitro. CBB, Coomassie Brilliant blue. (B) XEE was mock depleted or depleted (dep) with BuGZ antibody, and the BuGZ-depleted XEE was supplemented with the indicated purified proteins followed by xAurA immunoprecipitation and Western blotting analyses. r IgG, rabbit polyclonal IgG.
	Figure S1. Validation of cell synchronization. After releasing from the double thymidine block, the cells were collected at the indicated time, and cell lysates were analyzed by Western blotting with the indicated antibodies.
	Figure S2. xMCAK inhibition counteracts the effect of xBuGZ depletion in egg extracts. (A) XEE was mock depleted (control), was depleted (dep) of xBuGZ or xTPX2, or was treated with AurA inhibitor MLN8237 and then used for an AurA bead assay in the presence of unimmunized control IgG or a functional blocking xMCAK antibody (Ab). Bars, 10 μm. (B and C) The lengths of MT asters or numbers of bipolar spindles were quanti ed as described in Fig. 2. Error bars show SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. r IgG, rabbit polyclonal IgG.
	Figure S3. Tubulin binding to BuGZ involves the rst 58 amino acids, including the zinc nger sequence, and is reduced in the presence of EDTA. (A) Puri ed tubulin was incubated with puri ed proteins as indicated followed by pulldown using antibody to YFP as well as Western blotting and Coomassie Brilliant blue (CBB) staining. (B) Puri ed tubulin was incubated with puri ed GST or GST-xBuGZ(8–58) in the presence or absence of EDTA. For Western blotting, 0.1% of total reaction and 10% of total pulldowns were analyzed. For Coomassie blue staining, 1% of the reaction and 10% of total pulldowns were analyzed.

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