Hanley ML , Yoo TY , Sonnett M , Needleman DJ , Mitchison TJ .

F31 GM116451 NIGMS NIH HHS , R01 GM039565 NIGMS NIH HHS , R37 GM039565 NIGMS NIH HHS

	FIGURE 1:. Phosphatase inhibition activates AURKB and causes a change in the hydrodynamic properties of its associated protein complex. (A) Sequences of the activation loop of human and X. laevis AURKB and X. laevis AURKA. Differences are highlighted in red, and the key phosphorylated threonine is highlighted in turquoise. (B) Mitotic HSS was incubated with kinase inhibitors and okadaic acid. Equal reaction volumes were run on an SDS–PAGE gel and immunoblotted with antibodies against pT232-AURK (which recognizes AURKA, AURKB, and AURKC phosphorylated at the conserved threonine on the activation loop of the kinase), STMN, and α-tubulin. The blot was quantified by normalizing pAURKB intensity to α-tubulin intensity and set such that the lowest value of each replicate was the DMSO control (lanes where no intensity could be detected are marked as zero). The p value between the DMSO and okadaic acid samples is 0.014; n = 3. (C) Mitotic HSS was incubated with kinase inhibitor or DMSO for 25 min, followed by okadaic acid or DMSO for an additional 25 min. Equal volumes were sedimented on 5–40% sucrose gradients for 6 h at 237,000 × g and 4°C. The indicated fractions were separated on SDS–PAGE gels and immunoblotted with antibodies against INCENP and CDCA9. The identity of the higher–molecular weight band in higher-numbered INCENP blots is unknown but likely corresponds to a posttranslationally modified version of INCENP. Full blots with molecular weight markers are given in Supplemental Figure S1. (D) Sucrose gradient blots in C were quantified and normalized to the total amount of the indicated protein in the gradient. Error bars represent SD. The p values indicated by asterisks were calculated between the barasertib- and okadaic acid–treated samples in fractions 3 (INCENP 0.00046, CDCA9 0.0075) and 4 (INCENP 0.0057, CDCA9 0.0062); n = 4.
	FIGURE 2:. CPC hydrodynamic properties are similar in interphase and mitotic HSS. (A) Interphase HSS was prepared by cycling CSF X. laevis egg extract into interphase with calcium and then adding cycloheximide, followed by the high-speed spin. Equal amounts of interphase and mitotic HSS prepared from the same extract were blotted with an MPM2 (mitotic phosphoprotein monoclonal) antibody that recognizes many CDK1 phosphosites more prevalent in mitosis (Kuang et al., 1994; Field et al., 2014). Interphase extract was prepared more than five times and compared with mitotic HSS prepared from the same crude extract using MPM2 blots twice. (B) Sucrose gradients were run as in Figure 1. Mitotic and interphase HSS were prepared from the same extract. Fractions were immunoblotted with antibodies raised against INCENP and CDCA9 after SDS–PAGE. Full blots with molecular weight markers are given in Supplemental Figure S3. (C) Sucrose gradient blots from B were quantified as in Figure 1D. Error bars represent SD; n = 2. For fraction 4, significant p values comparing basal interphase HSS and interphase HSS dosed with 1 and 10 μM okadaic acid (OA) were calculated to be 0.011 (INCENP, 1 μM OA), 0.050 (INCENP, 10 μM OA), and 0.014 (CDCA9, 10 μM OA); n = 2–5. (D) Mitotic and interphase HSS samples prepared from the same extract were incubated with the indicated drugs as in Figure 1. The reactions were then blotted with antibodies against pT232-AURKB and α-tubulin, and pAURKB levels were quantified by normalizing to α-tubulin; n = 2.
	FIGURE 3:. Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with STLC and collected via mitotic shake-off, followed by washing with PBS. The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.
	FIGURE 4:. Fluorescence correlation spectroscopy of HeLa CDCA8-GFP-3xFLAG cells. Mitotic cells were identified and CPC localization to centrosomes confirmed by conventional imaging. The FCS measurement beam was directed into the cytoplasm near the edge of cells away from chromosomes to report on soluble CPC diffusion. (A) Example autocorrelation curves (top; black circles represent averages, and red and blue lines represent the fitted one- and two-component FCS models, respectively) and weighted residuals (bottom). (B) Ratio of amplitudes of slow- to fast-diffusing populations over time after drug treatment. Amplitudes are proportional to the number of molecules in a focal volume and molecular brightness squared. (C) Diffusion coefficients over time after drug treatment of the slow (left) and fast (right) populations in the presence of 1 µM nocodazole, with no further drug treatment (blue circles), or after treatment with only 250 nM okadaic acid (OA; green squares) or with both 250 nM OA and 250 nM barasertib (BA; red triangles). Each data point represents one mitotic cell, identified by observing round cell morphology and chromosomes in phase contrast, and averages were obtained by taking multiple measurements in each cell at each time point. Error bars represent the SD of the fitted parameters of the nonlinear regression.
	FIGURE 5:. NPM2 interacts with inactive CPC. (A) CPC proteins were immunoprecipitated from mitotic HSS and analyzed via quantitative mass spectrometry. Protein counts were normalized to IgG IPs and the target protein (INCENP or CDCA9). Ratios for each protein were generated by dividing reporter ion counts from barasertib-treated samples by counts from okadaic acid–treated samples. Proteins with a ratio >1 (gridlines on plot) indicate enrichment in barasertib-treated samples over okadaic acid–treated samples. Labeled proteins are those with ratios >1 in both CDCA8 and INCENP IPs. Size of bubbles is proportional to the measured amount of protein in X. laevis extract (Wuhr et al., 2014). Full lists of proteins and ratios are given in Supplemental Table S1. (B) Mitotic HSS was incubated with barasertib (BA), okadaic acid (OA), both, or neither as in Figure 1. Beads loaded with antibodies against X. laevis INCENP, CDCA9, and NPM2 were used for immunoprecipitations from treated mitotic HSS, and then the proteins on the beads were blotted with antibodies against INCENP and NPM2. Blots were quantified by dividing levels of the blotted protein by levels of the IP target protein. Fold change represents change in NPM2 levels (INCENP and CDCA9 IPs) or INCENP levels (NPM2 IP) after intensities were normalized by setting the DMSO lane to 1. Values of 0 indicate no detectable intensity. The p values between the BA and OA samples indicated by the asterisk are 0.029 (CDCA9 IP) and 0.043 (NPM2 IP); n = 2. Full blots, including molecular weight markers, are given in Supplemental Figure S6. (C) Crude X. laevis extract was cycled into interphase with the addition of calcium, and both mitotic and interphase extract were treated with drugs as in B. Beads loaded with antibody against CDCA9 were used for immunoprecipitation and immunoblotted for INCENP and NPM2. NPM2 blots were quantified as described, normalized to INCENP, and with the lane treated with both drugs set to 1. Full blot including molecular weight markers is given in Supplemental Figure S7. (D) The CPC was immunoprecipitated from mitotic HSS using the antibody raised against CDCA9. After four quick low-salt washes, beads were incubated in solutions of increasing salt for 5 min each. The proteins bound to the beads before and after the salt washes, as well as those in the entire volume of each salt wash, were separated via SDS–PAGE and protein levels analyzed by immunoblots of INCENP, NPM2, and actin. Protein levels were quantified as a percentage of the total of that protein. Error bars represent the SD; n = 2. Full blots including molecular weight markers are given in Supplemental Figure S8. (E) Sucrose gradients of barasertib (BA)- or okadaic acid (OA)–treated mitotic HSS were run as in Figure 1, except that fractions were one-third smaller. Every other fraction was immunoblotted for NPM2, INCENP, or CDCA9. NPM2 blots had 7.5 times less total protein concentration in each lane. NPM2 blots were quantified as in Figure 1. Error bars represent SD; n = 2. Full blots including molecular weight markers are given in Supplemental Figure S9. (F) Proposed model for phosphorylation-induced CPC hydrodynamic changes (CPC proteins: AURKB green, INCENP black line, CDCA8/9 orange, BIRC5 yellow). Pentameric or decameric NPM oligomers (blue) interact with the CPC when the CPC is inactive and unphosphorylated. This interaction may involve direct binding or be mediated by other proteins. When phosphatases are inhibited, NPMs dissociate and the CPC is activated.
	Figure S2: Mitotic HSS was diluted 1, 2, 3, 4, or 5-fold in S-CSF-XB-phosphate buffer, okadaic acid was added at a constant concentration, and the reactions were incubated at room temperature for 25 minutes. Reaction aliquots containing the same amount of HSS were immunoblotted with the antibody against the pT232-AURK epitope. The intensity of the samples was normalized to α-tubulin intensity and across biological replicates, setting the lowest value to one. Error bars represent standard deviations, and the p-value between 100% and 20% HSS is 0.0019, n=4.
	Figure S4. (A) Samples of mitotic or interphase crude X. laevis extract were immunoblotted using an antibody against MPM2 epitopes. (B) Droplets of interphase or mitotic extract (1 μL) from the same extract were placed into a room temperature mineral oil bath and observed via dark field microscopy for a period of approximately 45 minutes.
	Figure S10. Mitotic HSS was treated with no drug, barasertib, okadaic acid, or both, and then Dynabeads conjugated with antibody against CDCA9 were used to immunoprecipitate the CPC. The beads were then immunoblotted for INCENP and histone H2A.Z.
	FIGURE 1:. Phosphatase inhibition activates AURKB and causes a change in the hydrodynamic properties of its associated protein complex. (A) Sequences of the activation loop of human and X. laevis AURKB and X. laevis AURKA. Differences are highlighted in red, and the key phosphorylated threonine is highlighted in turquoise. (B) Mitotic HSS was incubated with kinase inhibitors and okadaic acid. Equal reaction volumes were run on an SDS–PAGE gel and immunoblotted with antibodies against pT232-AURK (which recognizes AURKA, AURKB, and AURKC phosphorylated at the conserved threonine on the activation loop of the kinase), STMN, and α-tubulin. The blot was quantified by normalizing pAURKB intensity to α-tubulin intensity and set such that the lowest value of each replicate was the DMSO control (lanes where no intensity could be detected are marked as zero). The p value between the DMSO and okadaic acid samples is 0.014; n = 3. (C) Mitotic HSS was incubated with kinase inhibitor or DMSO for 25 min, followed by okadaic acid or DMSO for an additional 25 min. Equal volumes were sedimented on 5–40% sucrose gradients for 6 h at 237,000 × g and 4°C. The indicated fractions were separated on SDS–PAGE gels and immunoblotted with antibodies against INCENP and CDCA9. The identity of the higher–molecular weight band in higher-numbered INCENP blots is unknown but likely corresponds to a posttranslationally modified version of INCENP. Full blots with molecular weight markers are given in Supplemental Figure S1. (D) Sucrose gradient blots in C were quantified and normalized to the total amount of the indicated protein in the gradient. Error bars represent SD. The p values indicated by asterisks were calculated between the barasertib- and okadaic acid–treated samples in fractions 3 (INCENP 0.00046, CDCA9 0.0075) and 4 (INCENP 0.0057, CDCA9 0.0062); n = 4.
	FIGURE 2:. CPC hydrodynamic properties are similar in interphase and mitotic HSS. (A) Interphase HSS was prepared by cycling CSF X. laevis egg extract into interphase with calcium and then adding cycloheximide, followed by the high-speed spin. Equal amounts of interphase and mitotic HSS prepared from the same extract were blotted with an MPM2 (mitotic phosphoprotein monoclonal) antibody that recognizes many CDK1 phosphosites more prevalent in mitosis (Kuang et al., 1994; Field et al., 2014). Interphase extract was prepared more than five times and compared with mitotic HSS prepared from the same crude extract using MPM2 blots twice. (B) Sucrose gradients were run as in Figure 1. Mitotic and interphase HSS were prepared from the same extract. Fractions were immunoblotted with antibodies raised against INCENP and CDCA9 after SDS–PAGE. Full blots with molecular weight markers are given in Supplemental Figure S3. (C) Sucrose gradient blots from B were quantified as in Figure 1D. Error bars represent SD; n = 2. For fraction 4, significant p values comparing basal interphase HSS and interphase HSS dosed with 1 and 10 μM okadaic acid (OA) were calculated to be 0.011 (INCENP, 1 μM OA), 0.050 (INCENP, 10 μM OA), and 0.014 (CDCA9, 10 μM OA); n = 2–5. (D) Mitotic and interphase HSS samples prepared from the same extract were incubated with the indicated drugs as in Figure 1. The reactions were then blotted with antibodies against pT232-AURKB and α-tubulin, and pAURKB levels were quantified by normalizing to α-tubulin; n = 2.
	FIGURE 3:. Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with STLC and collected via mitotic shake-off, followed by washing with PBS. The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.
	FIGURE 4:. Fluorescence correlation spectroscopy of HeLa CDCA8-GFP-3xFLAG cells. Mitotic cells were identified and CPC localization to centrosomes confirmed by conventional imaging. The FCS measurement beam was directed into the cytoplasm near the edge of cells away from chromosomes to report on soluble CPC diffusion. (A) Example autocorrelation curves (top; black circles represent averages, and red and blue lines represent the fitted one- and two-component FCS models, respectively) and weighted residuals (bottom). (B) Ratio of amplitudes of slow- to fast-diffusing populations over time after drug treatment. Amplitudes are proportional to the number of molecules in a focal volume and molecular brightness squared. (C) Diffusion coefficients over time after drug treatment of the slow (left) and fast (right) populations in the presence of 1 µM nocodazole, with no further drug treatment (blue circles), or after treatment with only 250 nM okadaic acid (OA; green squares) or with both 250 nM OA and 250 nM barasertib (BA; red triangles). Each data point represents one mitotic cell, identified by observing round cell morphology and chromosomes in phase contrast, and averages were obtained by taking multiple measurements in each cell at each time point. Error bars represent the SD of the fitted parameters of the nonlinear regression.
	FIGURE 5:. NPM2 interacts with inactive CPC. (A) CPC proteins were immunoprecipitated from mitotic HSS and analyzed via quantitative mass spectrometry. Protein counts were normalized to IgG IPs and the target protein (INCENP or CDCA9). Ratios for each protein were generated by dividing reporter ion counts from barasertib-treated samples by counts from okadaic acid–treated samples. Proteins with a ratio >1 (gridlines on plot) indicate enrichment in barasertib-treated samples over okadaic acid–treated samples. Labeled proteins are those with ratios >1 in both CDCA8 and INCENP IPs. Size of bubbles is proportional to the measured amount of protein in X. laevis extract (Wuhr et al., 2014). Full lists of proteins and ratios are given in Supplemental Table S1. (B) Mitotic HSS was incubated with barasertib (BA), okadaic acid (OA), both, or neither as in Figure 1. Beads loaded with antibodies against X. laevis INCENP, CDCA9, and NPM2 were used for immunoprecipitations from treated mitotic HSS, and then the proteins on the beads were blotted with antibodies against INCENP and NPM2. Blots were quantified by dividing levels of the blotted protein by levels of the IP target protein. Fold change represents change in NPM2 levels (INCENP and CDCA9 IPs) or INCENP levels (NPM2 IP) after intensities were normalized by setting the DMSO lane to 1. Values of 0 indicate no detectable intensity. The p values between the BA and OA samples indicated by the asterisk are 0.029 (CDCA9 IP) and 0.043 (NPM2 IP); n = 2. Full blots, including molecular weight markers, are given in Supplemental Figure S6. (C) Crude X. laevis extract was cycled into interphase with the addition of calcium, and both mitotic and interphase extract were treated with drugs as in B. Beads loaded with antibody against CDCA9 were used for immunoprecipitation and immunoblotted for INCENP and NPM2. NPM2 blots were quantified as described, normalized to INCENP, and with the lane treated with both drugs set to 1. Full blot including molecular weight markers is given in Supplemental Figure S7. (D) The CPC was immunoprecipitated from mitotic HSS using the antibody raised against CDCA9. After four quick low-salt washes, beads were incubated in solutions of increasing salt for 5 min each. The proteins bound to the beads before and after the salt washes, as well as those in the entire volume of each salt wash, were separated via SDS–PAGE and protein levels analyzed by immunoblots of INCENP, NPM2, and actin. Protein levels were quantified as a percentage of the total of that protein. Error bars represent the SD; n = 2. Full blots including molecular weight markers are given in Supplemental Figure S8. (E) Sucrose gradients of barasertib (BA)- or okadaic acid (OA)–treated mitotic HSS were run as in Figure 1, except that fractions were one-third smaller. Every other fraction was immunoblotted for NPM2, INCENP, or CDCA9. NPM2 blots had 7.5 times less total protein concentration in each lane. NPM2 blots were quantified as in Figure 1. Error bars represent SD; n = 2. Full blots including molecular weight markers are given in Supplemental Figure S9. (F) Proposed model for phosphorylation-induced CPC hydrodynamic changes (CPC proteins: AURKB green, INCENP black line, CDCA8/9 orange, BIRC5 yellow). Pentameric or decameric NPM oligomers (blue) interact with the CPC when the CPC is inactive and unphosphorylated. This interaction may involve direct binding or be mediated by other proteins. When phosphatases are inhibited, NPMs dissociate and the CPC is activated.

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