Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multi-protein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo . HsMis13 is a cognate substrate of Aurora B and the phosphorylation sites were mapped to Ser100 and Ser109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13, failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

The kinetochore is a super-molecular complex assembled at each centromere in eukaryotes. It provides a chromosomal attachment point for the mitotic spindle, linking the chromosome to the microtubules and functions in initiating, controlling and monitoring the movements of chromosomes during mitosis. The kinetochore of animal cells contains two functional domains: the inner kinetochore which is tightly and persistently associated with centromeric DNA sequences throughout the cell cycle, and the outer kinetochore that is composed of many dynamic protein complexes that interact with microtubules only during mitosis. The stable propagation of eukaryotic cells requires each chromosome to be accurately duplicated and faithfully segregated. During mitosis, attaching, positioning and bi-orientating kinetochores with the spindle microtubules play critical roles in chromosome segregation and genomic stability (see refs. [1][2]. Mitosis is orchestrated by signaling cascades that coordinate mitotic processes and ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase Cdk1. In addition to the master mitotic kinase Cdk1, three other protein serine/threonine kinase families are also involved, including the Polo kinases, Aurora kinases, and the NEK (NIMA-related kinases) (e.g., [3][4]. Recent studies have demonstrated the involvement of NEK kinase in stabilization of the kinetochore-microtubule attachment (e.g., 5) and the critical role of Aurora B kinase in kinetochore bi-orientation (e.g., 6). While the mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation control, the molecular details remain elusive.
Early studies to isolate temperature-sensitive fission yeast mutants that displayed high loss rates of mini-chromosomes at permissive or semi-permissive temperatures had lead Yanagida and associates to identify prometaphase and 11 hours to synchronize at telophase. In some cases, 100 nM nocodazole was added to cell culture for 18 hours to synchronize cells in prometaphase.

cDNA construction
To generate GFP-tagged full-length HsMis13, PCR amplified HsMis13 cDNA was digested with BgLII and SalI and then cloned into pEGFP-C3 vector (Clontech). The bacterial expression constructs of HsMis13 were cloned into pGEX-6P-1 vector (Amersham Biosciences) with SmaI digestion. FLAG-tagged HsMis13 cDNA was cloned by inserting the PCR product into the p3XFLAG-myc-CMV-24 vector (Sigma) with EcoRV digestion.
GFP-tagged non-phosphorylatable and phosphormimetic HsMis13 mutants were created by standard PCR methods as described previously (e.g., 15). All constructs were sequenced in full.

Recombinant protein expression
Human HsMis13 was expressed in bacteria as a GST fusion protein. Briefly, 500 ml LB media was inoculated with bacteria Rosetta (DE3) pLysS transformed with GST-HsMis13. The protein expression was initiated by addition of 0.2 mM IPTG and incubation at 16 °C for 12 hours. Bacteria were then collected by centrifugation and resuspended in phosphate buffered saline (PBS) containing a protease inhibitor cocktail (Sigma) followed by sonication and clarification. The GST fusion protein in bacteria in the soluble fraction was purified by using glutathione-agarose chromatography according to manufacturer's protocol.

Antibodies
Affinity purification of CENP-E rabbit antibody was described previously (16). Mouse monoclonal antibody to Nuf2 was purchased from Abcam (UK) and diluted at 1:1000 for western blot and 1:300 for immunofluorescence. Mouse monoclonal antibody to Hec1 was purchased from Abcam and diluted at 1:1,000 for western blot and 1:500 for immnofluorescence. Mouse monoclonal antibodies to GST was purchased from Cell Signaling (Beverly, MA), and used at a 1:1000 dilution for western blot. Mouse monoclonal antibody to GFP was obtained from BD Biosciences (Palo Alto, CA).
Purified His-HsMis13 from inclusion body was used to immunize rabbit and Balb/C mice according to the general protocol. The purified IgG was obtained by affinity purification using an HsMis13 affinity matrix based on CNBr-activated agarose beads (Sigma).
In some instances, HeLa cells were synchronized at prophase and telophase as described above. The cells were then lysed using lysis buffer (0.5% NP-40, 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide, 1 mM PMSF was added prior to use). HsMis13 antibody was incubated with protein A/G beads. Protein bound protein A/G beads were washed 3 times by wash buffer (0.1% NP-40, 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide, PMSF was added just before use) to prepare for the immunoprecipitation experiment.
These antibodybound A/G beads were added to cell lysate and incubated for 4 hours at 4 °C. After incubation, beads were washed 4 times with wash buffer and once with PBS. Samples were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membrane to perform western blot.
For immunoprecipitation of transiently transfected cells, HeLa cells were generally collected 42 hours after transfection of GFP-HsMis13 and GFP-HsMis13 AA , which included a 18-hour nocodazole synchronization. Cellular proteins were solubilized in lysis buffer and clarified by centrifugation. GFP-HsMis13 proteins were precipitated using a rabbit anti-GFP antibody bound to protein-A/G beads (Pierce Chemical, IL). Beads were washed five times with lysis buffer and then boiled in protein sample buffer for 2 min. After SDS-PAGE, proteins were transferred to nitrocellulose membrane. The membrane was probed with antibodies against GFP and phospho-serine (Sigma) as described (5).

Transfection and immunofluorescence
Cells were transfected with siRNA or GFP tagged plasmids in a 24-well plate by Oligofectamine reagent and Lipofectamine 2000 (Invitrogen, CA), respectively, according to the manufacture's manuals.
For immunofluorescence, HeLa cells were seeded onto sterile, acid-treated 12 mm coverslips in 24-well plates (Corning Glass Works, Corning, New York). Double thymidine blocked and released HeLa cells were transfected with 1 μl lipofectamine 2000 pre-mixed with 1 μg various plasmids as described above. In general, 36-48 h after transfection, HeLa cells were rinsed for 1 min with PHEM buffer (100 mM PIPES, 20 mM HEPES, pH 6.9, 5 mM EGTA, 2 mM MgCl 2 and 4 M glycerol) and were permeabilized for 1 min with PHEM plus 0.1% Triton X-100. Extracted cells were then fixed in freshly prepared 4% paraformaldehyde in PHEM, and rinsed three times in PBS. Cells on the coverslips were blocked with 0.05% Tween 20 in PBS (TPBS) with 1% BSA (Sigma). These cells were incubated with various primary antibodies in a humidified chamber for 1 h and then washed three times in TPBS. Primary antibodies were visualized using FITC-conjugated goat anti-mouse IgG or rhodomine-conjugated goat anti-rabbit IgG respectively. DNA was stained with DAPI (Sigma Chemicals).

In vitro kinase assay
His-tagged Aurora-B kinase was expressed in bacteria and purified by Ni 2+ -NTA beads. The kinase reactions were performed in 40 μl 1× kinase buffer (25 mM HEPES, pH 7.2, 5 mM MgSO 4 , 1 mM DTT, 50 mM NaCl, 2 mM EGTA) containing 1 μl eluted Aurora-B kinase, 5 μl GST beads bound GST-Mis13, 5 μCi γ-32 P-ATP and 50 μM ATP. The mixtures were incubated at 30 °C for 30 min. The reactions were stopped with 2× SDS sample buffer and separated by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue, dried, and the 32 P incorporation into HsMis13 proteins was quantified by a PhosphorImager (Amersham Biosciences). The specific incorporation of 32 P into wild type HsMis13 and non-phosphorylatable HsMis13 S100/109A was normalized to protein levels and expressed as relative activity to the wild type protein.

Small inhibitory RNA (SiRNA)
For the siRNA studies, the 21-mer of siRNA duplexes against HsMis13 and Aurora B were synthesized by Dharmacon Research Inc. (Lafayette, CO) as previously described (11,13,18). As a control, either a duplex targeting cyclophilin or scramble sequence was used (11).
The transfection efficiency was judged based on the uptake of the fluorescein isothiocyanate-conjugated oligonucleotides whereas the efficiency of siRNA-mediated protein suppression was judged by Western blotting analysis (e.g., 11). After trial experiments using a series of concentration and time course assay, treatment of 150 nM for 36 hours was finally selected as the most efficient conditions for repressing target proteins.

Fluorescence intensity quantification and kinetochore distance measurement
Fluorescence intensity of kinetochore protein labeling was measured by using the confocal microscope LSM510 NLO (Carl Zeiss) scan head mounted transversely to an inverted microscope (Axiovert 200; Carl Zeiss) with a 100 × 1.3 NA PlanApo objective. The images from double labeling were collected using a dichroic filter set with Zeiss image processing software (LSM 5, Carl Zeiss). The distance between sister kinetochores marked with ACA was measured as the distance between the peak fluorescence as previously described (17).
The quantification of the level of kinetochoreassociated protein was conducted as described by Johnson et al., (18) and more recently by Liu et al., (24). In brief, the average pixel intensities from at least 100 kinetochore pairs from five cells were measured and background pixel intensities subtracted. The pixel intensities at each kinetochore pair were then normalized against ACA pixel values to account for any variations in staining or image acquisition. The values of specific siRNA-treated cells were then plotted as a percentage of the values obtained from cells transfected with a control siRNA duplex.

HsMis13 is a novel Aurora B-binding partner
To delineate the molecular mechanism underlying Aurora B regulation in kinetochore protein-protein interaction networks, we immobilized bacterially recombinant kinetochore proteins onto a nitrocellulous membrane to conduct a quick search for Aurora B binding proteins using a "high-content" far-western assay (5,14). The Aurora B-binding activity was then detected by an anti-Aurora B monoclonal antibody. One advantage of such a "high-content" assay is to screen a large number of potential interacting proteins in parallel yet avoid initial intensive labor spent on purifying large quantitites of proteins required for pull-down assays. This assay has detected the binding activity of several known Aurora B-interacting proteins, such as Hec1, validating the sensitivity of such an assay (Supplemental Fig. 1). This assay is very specific as Aurora B was never been found to be associated with GST tag, MBP tag, BSA. Interestingly, Aurora B was found to associate with HsMis13 immobilized onto the membrane (Supplemental Fig. 1; C1), suggesting a potential interaction between Aurora B kinase and HsMis13 protein.
To examine whether HsMis13 forms a cognate complex with Aurora B in cells, we carried out an immunoprecipitation using mitotic lysates from 293T cells transiently transfected to express FLAG-HsMis13 and GFP-Aurora B (both wild type and kinase death mutant). As shown in Figure 1A, western blot using GFP antibody confirmed that Aurora B was pulled down by FLAG-HsMis13 (lanes 5-6; upper blot). However, a greater amount of wild type Aurora B was recovered in FLAG-HsMis13 immunoprecipitates compared to that of kinase-death Aurora B mutant. No GFP tag was precipitated with FLAG-HsMis13 (lane 4). Thus, we conclude that HsMis13 interacts with Aurora B in vivo.
If HsMis13 is a cognate binding partner of Aurora B, they should co-distribute to kinetochore of mitotic cells. To this end, we performed an immunocytochemical staining in which an anti-HsMis13 rabbit antibody and an anti-Aurora B mouse antibody were employed to mark their kinetochore distribution. As shown in Figure 1B, Aurora B displayed a typical inner kinetochore distribution from prophase to metaphase in HeLa cells (arrows; c, g, and k) while HsMis13 exhibited a typical pair of separated kinetochore spots (arrowheads; a, e, and i). The merged image confirmed their relative co-distribution at the centromere (arrow; d, h and l).

HsMis13 is a novel substrate of Aurora B
Since HsMis13 binds to Aurora B in vivo, we sought to test if HsMis13 is a substrate of Aurora B. Our computational analysis suggests that Ser 100 and Ser 109 are potential substrates of Aurora B (19), which are conserved among vertebrate HsMis13 proteins. To test whether Ser 100 and Ser 109 are substrates of Aurora B, we performed in vitro phosphorylation on recombinant GST-HsMis13 fusion proteins, including both wild type HsMis13 and non-phosphorylatable HsMis13 mutants in which Ser 100 and Ser 109 were both replaced by alanine (HsMis13 S100/109A ). Both GST fusion proteins, wild type and mutant HsMis13 S100/109A , migrated to about the predicted 68 kDa as shown in Figure 1C. Incubation of the fusion proteins with [ 32 P]-ATP and His-Aurora B resulted in the incorporation of 32 P into wild type but not HsMis13 S100/109A mutant ( Figure 1C, upper panel). This Aurora B-mediated phosphorylation is specific, since incubation of HsMis13 with [ 32 P]-ATP in the absence of Aurora B resulted in no detectable incorporation of radioactivity into the wild type protein (data not shown). The specific incorporation of 32P into wild type protein versus non-phosphorylatable mutant was quantified and shown in Fig. 1D, which show that only background level 32 P was incorporated into non-phosphorylatable HsMis13 protein in the presence of active Aurora B kinase. Thus, we conclude that both Ser100 and Ser109 on HsMis13 are substrates of Aurora B in vitro.
If HsMis13 is a substrate of Aurora B in vivo, suppression of Aurora B kinase activity using chemical inhibitors, such as VX-680 (20), would alter endogenous HsMis13 phosphorylation. Indeed, suppression of Aurora B using VX-680 reduced serine phosphorylation of HsMis13 immunopurified from mitotic HeLa cells ( Figure 1E), suggesting that HsMis13 is a cognate substrate of Aurora B in mitosis.
To validate if Ser100 and Ser109 are Aurora B substrates in vivo, we conducted an anti-GFP immunoprecipitation of nocodazole-arrested mitotic cell lysates from HeLa cells transiently transfected to express GFP-HsMis13 and GFP-HsMis13 S100/109A proteins. Importantly, GFP-HsMis13 protein but not GFP-HsMis13 S100/109A mutant protein contains serine phosphorylation judging by anti-phospho-serine western blotting analysis ( Figure 1F). Thus, we conclude that Ser100 and Ser109 of HsMis13 are endogenous substrates of Aurora B in mitotic cells.

Aurora B kinase activity controls the kinetochore localization of HsMis13
To investigate the possible influence of Aurora B on the localization of HsMis13 to the kinetochore, we introduced siRNA oligonucleotide duplexes to Aurora B by transfection into HeLa cells. Trial experiments revealed that treatment of HeLa cells with 150 nM siRNA for 36 hours produced an optimal suppression of the target protein. As shown in Figure 2A, Western blot with an anti-Aurora B antibody revealed that the siRNA oligonucleotide caused remarkable suppression of Aurora-B protein levels at 36 h. This suppression is relatively specific as it did not alter the levels of other proteins such as tubulin and HsMis13.
Next, we examined the effect of repressing Aurora B on the localization of HsMis13 to the kinetochore. HeLa cells were subcultured on coverslips in 24-well plates, and transfected with siRNA. In control scramble siRNA-transfected cultures, Aurora B and HsMis13 were co-distributed to the kinetochore of prometaphase and metaphase cells ( Figure 2B; a-h). In cells in which Aurora B had been suppressed, the levels of kinetochore-bound HsMis13 appeared reduced ( Figure  2B; a'-h'). Quantification of normalized pixel intensities shows that, when Aurora B was reduced to less than 11% of its control value, HsMis13 levels were reduced to ~29%, indicating that Aurora B is required for efficient kinetochore localization of HsMis13 ( Figure  2D) Since Aurora B and HsMis13 form a stable complex, failure of HsMis13 localization to the kinetochore could be mediated by their physical interactions. To assess whether Aurora B kinase activity, rather than its physical interaction with HsMis13, controls the kinetochore localization of HsMis13, we sought to use Aurora B inhibitor VX-680 and hersperadin to suppress Aurora kinase activity and then assess the effect of Aurora B inhibition on the localization of HsMis13 to the kinetochore. As shown in Figure 2C (c'), 1 µM VX-680 almost completely suppressed histone 3 phosphorylation on Ser10, an endogenous substrate of Aurora B, consistent with the literature (20). Examination of the same cell treated with VX-680 revealed that HsMis13 localization to the kinetochore was minimized ( Figure 2C, a').
Quantification of normalized pixel intensities shows that, when Aurora B activity was reduced to less than 10% of its control value as judged by histone Ser10 phosphorylation, HsMis13 levels were reduced to ~31% ( Figure 2D). Experimentation with another Aurora B kinase inhibitor hersperadin gave essentially the same outcome (data not shown). Thus, we conclude that Aurora B kinase activity controls the localization of HsMis13 to the kinetochore.

HsMis13 exhibits a cell cycle-dependent distribution to kinetochore
The cell cycle-regulated spatiotemporal dynamics of Aurora B propelled us to examine the distribution profile of HsMis13 in mitotic cells. Western blotting analyses of synchronized HeLa cell lysates show that the HsMis13 protein expression level is relatively constant during cell cycle while CENP-E and cyclin B levels exhibit a typical cyclic wave ( Figure 3A). We next examined the spatiotemporal distribution of HsMis13 in HeLa cells. The ACA immunoflorescence study shows a typical labeling of centromere in interphase nucleus and kinetochore of mitotic cells ( Figure 3B, green). However, HsMis13 began to appear on the kinetochore marked by ACA staining in prophase and remains associated with kinetochore until anaphase B.
In telophase, ACA staining remains associated with kinetochore while HsMis13 is no longer associated with kinetochore ( Figure 3B; g), suggesting that HsMis13 is assembled onto the centromere during interphase and released from centromere at telophase. To test if HsMis13 localization to kinetochore is correlated with its phosphorylation by Aurora B, we isolated the HsMis13 from prophase and telophase lysates from thymidine synchronized and released HeLa cells. Consistent with our hypothesis, the HsMis13 isolated from prophase, but not telophase cells, contains a phospho-serine epitope ( Figure 3C).
The lack of Ser10 phosphorylation of histone 3 indicates that Aurora B activity is minimized at the kinetochore of telophase cells ( Figure 3C; left). Thus, we reason that cell cycle regulated association of HsMis13 with the kinetochore is a function of Aurora B phosphorylation.

Aurora B-mediated phosphorylation determines HsMis13 association with kinetochore
To directly examine the role of Aurora B-mediated phosphorylation in HsMis13 localization to the kinetochore, we generated non-phosphorylatable and phosphomimetic HsMis13 mutations and transiently transfected HeLa cells with GFP-tagged mutant plasmids. Western blotting analysis carried out using transfected HeLa cell preparations showed that the exogenously expressed GFP-HsMis13 protein was about twice the level of endogenous HsMis13 ( Figure 4A). Given transfection efficiency of 55~60%, the actual expression level of GFP-HsMis13 in positively transfected cells is about 3-folds higher than that of endogenous protein.
The subcellular localization of the exogenously expressed GFP-HsMis13 constructs was compared with that of HsMis12 by fluorescence microscopy. The transfected cells were triple-stained for GFP-HsMis13 using a monoclonal GFP antibody (green) and counter-stained for HsMis12 (red) and DNA (blue). Figure 4B shows the confocal image from wild type GFP-HsMis13-transfected cells. Similar to what has been noted in endogenous HsMis13 distribution, GFP-tagged HsMis13 distributes to the kinetochore of prophase cells ( Figure 4B; b), and is superimposed onto that of HsMis12 when the two channels are merged ( Figure 4B; d).
Our examination of GFP-HsMis13 S100/109A transfected cells revealed that non-phosphorylatable HsMis13 fails to localize to the kinetochore while the kinetochore distribution of HsMis12 remains ( Figure  4C). As predicted, phosphomimetic HsMis13 is localized to the kinetochore in a pattern similar to that of wild type HsMis13 ( Figure 4D). Thus, we conclude that Aurora B-mediated phosphorylation of HsMis13 at Ser100 and Ser109 controls the localization of HsMis13 to the kinetochore.

Aurora B-mediated phosphorylation of HsMis13 is essential for kinetochore assembly
HsMis13 is an important component of HsMis12 centromere core complex and essential for outer kinetochore protein complex assembly (e.g., refs. 11-13). However, it has remained elusive as to how the HsMis12 complex assembly is regulated and whether this determines the outer kinetochore protein complex assembly. To investigate the possible influence of HsMis13 on the localization of outer kinetochore protein complex assembly, we introduced siRNA oligonucleotide duplexes into HsMis13 by transfection into HeLa cells. As shown in Figure 5A, Western blot with an anti-HsMis13 antibody revealed that the siRNA oligonucleotide caused remarkable suppression of HsMis13 protein level at 48 h. This suppression is relatively specific as it did not alter the levels of other proteins such as Aurora B, CENP-E, and CENP-F etc.
Since HsMis13 and HsMis12 form a stable and evolutionarily conserved kinetochore core complex, we next examined the effect of repressing HsMis13 on the localization of CENP-E and CENP-F to the kinetochore. HeLa cells were subcultured on coverslips in 24-well plates, and transfected with siRNA. In control cultures, HsMis13 localized with ACA at the prometaphase kinetochores ( Figure 5B control). In cells in which HsMis13 had been suppressed, the levels of kinetochore-bound CENP-E appeared reduced ( Figure 5B; e'-h'). Quantification of normalized pixel intensities shows that, when HsMis13 was reduced to less than 10% of its control value, CENP-E level were reduced to ~37%, indicating that HsMis13 is required for efficient kinetochore localization of CENP-E, consistent with previous studies (14).
However, in these HsMis13-repressed cells, the levels of ACA and CENP-F detectable at kinetochores appeared largely unaffected ( Figure 5B; c' and k'). Quantification of normalized pixel intensities shows that, when HsMis13 was reduced to less than 10% of its control value, CENP-F levels were modestly reduced to ~69% of its control, suggesting that CENP-F location to kinetochore is also dependent on HsMis13, but to a less degree.
In cells in which HsMis13 had been suppressed, the levels of kinetochore-bound HsHec1 and HsNuf2 were also reduced. Quantification of normalized pixel intensities shows that, when HsMis13 was reduced to less than 10% of its control value, HsNuf2 levels were reduced to ~21% while Hec1 levels were reduced to ~23% ( Figure 5C), indicating that HsMis13 is required for efficient kinetochore localization of Ndc80 complex, consistent with the requirement of HsMis12 complex for faithful assembly of Ndc80 complex to outer kinetochore (e.g., ref. 13).
We then examined the effect of Aurora B-mediated phosphorylation of HsMis13 in kinetochore localization of CENP-E.
Previous studies show that Aurora B kinase activity controls the assembly of CENP-E to the kinetochore (e.g., refs. [21][22]. However, it is unclear how Aurora B signaling pathway controls CENP-E localization. If Aurora B-mediated phosphorylation of HsMis13 is essential for kinetochore assembly, expression of phosphomimetic mutant HsMis13 should restore the localization of CENP-E to kinetochore in the absence of Aurora B kinase activity. To test this hypothesis, we transfected HeLa cells with wild type and phosphomimetic mutant HsMis13 plasmids followed by treatment of VX-680 to inhibit Aurora B kinase activity. Consistent with what was observed for endogenous HsMis13 distribution in VX-680-treated cells ( Figure 2C, a'), GFP-HsMis13 fails to localize to the kinetochore in the presence of Aurora B inhibition ( Figure 5D, b) while HsMis12 remains kinetochore associated ( Figure 5D, a). As predicted, the phosphomimetic mutant HsMis13 co-distributes with HsMis12 to the kinetochore in the presence of VX-680 ( Figure 5D; b), confirming the role of Aurora B-mediated phosphorylation in the control of HsMis13 assembly to the kinetochore.
We next examined whether phosphomimetic mutant HsMis13 can restore CENP-E localization to the kinetochore in the presence of Aurora inhibition. Consistent with our prediction, CENP-E co-distributes with phosphomimetic HsMis13 S100/109E mutant to kinetochore in the presence of VX-680 ( Figure 5E; a) while little CENP-E appears localized to the kinetochore in GFP-HsMis13-expressing cells in the presence of VX-680 (quantitative analyses in Figure  5F).
As shown in Figure 5F (open bar), quantification of normalized pixel intensities shows that the kinetochore-bound CENP-E level, in the phosphomimetic HsMis13 S100/109E -expressing cells, is increased from ~27% to ~83% of its control value (in the absence of VX-680). Thus, we conclude that Aurora B-mediated phosphorylation of HsMis13 is required for the kinetochore localization of CENP-E.
Our recent study demonstrates that Nuf2 interacts with and specifies CENP-E localization to the kinetochore (24). Since CENP-E localization to kinetochore is a function of HsMis13 phosphorylation, we hypothesize that assembly of Nuf2 to the kinetochore is also a function of HsMis13 phosphorylation. As predicted, our quantification shows that the assembly of Nuf2 to the kinetochore, in the phosphomimetic HsMis13 S100/109E -expressing cells, is also increased (from ~21% to ~67% of its control value; Figure 5F, blue bar), consistent with the fact that Nuf2 mediates CENP-E localization to the kinetochore.

Phosphorylation of HsMis13 is essential for assembly a stable microtubule-kinetochore attachment
Previous studies have established that CENP-E and the HsNUF2-HEC1 complex are essential for stabilizing microtubule-kinetochore attachments (e.g., refs. 14, 18). Distance between the sister kinetochores marked by ACA has been used as an accurate reporter for judging the tension developed across the kinetochore pair (e.g. ref. 18). In this case, shortened distance often reflects aberrant microtubule attachment to the kinetochore, in which less tension is developed across the sister kinetochore. To test the functional activity of microtubule capturing in the cells suppressed Aurora B but expressing phosphomimetic HsMis13 S100/109E mutant, we measured this distance in 200 kinetochore pairs in which both kinetochores were in the same focal plane in both siRNA-treated cells and control cells ( Figure 6B).
As shown in Figure 6A, suppression of Aurora B results in errors in chromosome alignment at the equator, which is consistent with previous reports (e.g. Refs. 21,23). Control kinetochores exhibited a separation of 1.69 ± 0.14 µm whereas the mean distance between kinetochores was 1.27 ± 0.11 µm in Aurora B-suppressed cells. Significantly, expression of phosphomimetic HsMis13 S100/109E mutant increases inter-kinetochore distance (1.53 ± 0.17 µm) in the presence of VX-680. Withdrawal of VX-680 from HsMis13 S100/109E -expressing cells did not significantly extend the inter-kinetochore distance (1.67 ± 0.17 µm). Thus, we conclude that phosphorylation of HsMis13 facilitates the assembly of outer kinetochore which is essential for stable microtubule-kinetochore attachment.

DISCUSSION
The Mis12 complex is composed of four proteins: Mis12, Mis13, Mis14, and Mis15. This complex is an essential kinetochore core component highly conserved across species, with a crucial role in kinetochore assembly and proper chromosome segregation during mitosis (11,17,18). Our study revealed an important interaction between Aurora B and HsMis13, which is essential for kinetochore assembly and kinetochore-microtubule association.
Previous studies have established that the localization of the Ndc80 complex is exterior to the inner kinetochore proteins such as HsMis12 complex (11,13), and the Ndc80 complex localizes to the interior of CENP-E (e.g., refs. [23][24]. While the Ndc80 complex is indispensable for establishing kinetochore-microtubule attachments, the HsMis12 complex is linked to heterchromatin via binding to HP1 (e.g., 11). Thus, the Ndc80 complex is postulated to link microtubule-binding proteins and chromatin-bound centromere core proteins. It has been reported that the main function of the Ndc80 complex is to stabilize the microtubule-kinetochore attachment, as cells lacking Nuf2 or Hec1 often carry unstable spindle microtubules (23). Our recent finding of CENP-E-Nuf2 interaction provides a novel link between spindle microtubules and the kinetochore core complex via mitotic kinesin CENP-E (24). Consistent with this multi-protein complex architecture, suppression of any component of HsMis12 diminishes the association of CENP-E with the kinetochore (e.g, 13 and this study). It would be of great interest to delineate the precise molecular interaction underlying the HsMis12-Ndc80-CENP-E subcomplex assembly in vitro and examine how perturbation of such an interaction alters the plasticity of kinetochore assembly and chromosome segregation by a combination of nanometer-resolution distribution of single molecules with photoactivatable fluorescent proteins in living dividing cells (25).
The interaction between spindle microtubules and kinetochore is central to chromosome segregation and stability in mitosis.
The four-layer kinetochore ultra-structure is reflected by a step-wise assembly of kinetochore components from the chromatin to the outmost fiberous corona. Following breakdown of the nuclear envelope, astral microtubules emanate from centrioles and pass though gaps in the envelope coming in close proximity to newly condensing chromosomes. Using immunoelectron microscopy, CENP-E is found at developing kinetochores adjacent to microtubules at this early stage of mitosis (11). CENP-E is located at outer kinetochore surface during chromosome bi-orientation although the kinetochore remains morphologically immature. Despite the ultra-resolution analysis of CENP-E molecular dynamics in mitosis and recent identification of Ndc80 as a key structural determinant for CENP-E localization to kinetochore (24), the molecular architect of mammalian kinetochore has remained elusive. Recent studies have established the role of CENP-A as a fundamental determinant of centromere specification (26), and suggested a potential role of Mis12 complex as a potential link between CENP-A complex and kinetochore assembly foci (11,13). However, it was unclear how the individual subunit of the conserved four-subunit Mis12 complex is assembled onto the centromere.
Our finding that Aurora B phosphorylates HsMis13 and such phosphorylation controls HsMis13 assembly to kinetochore suggest that the complex is assembled at the kinetochore as HsMis12 is located to centromere prior to HsMis13. Interestingly, the localization of HsMis12 to the centromere is independent of Aurora B kinase. It would be important and necessary to examine whether and how Aurora B regulates the four-subunit complex assembly in vitro and in vivo.
One interesting phenotype shown in Figure 5E demonstrates that expressing the phosphomimetic HsMis13 S100/109E mutant in HeLa cells with diminished Aurora B kinase activity restores the kinetochore localization of CENP-E but fails to correct all misaligned chromosomes, indicating that the molecular regulation of their kinetochore components such as Ndc80 and MCAK by Aurora B is essential for faithful chromosome segregation. Besides its critical role in kinetochore assembly, Aurora kinase also governs kinetochore-microtubule interaction by correcting aberrant attachments (27). Consistent with this notion, recent studies show that phosphorylation of Hec1 by Aurora B is essential to orchestrate a dynamic and faithful kinetochore-microtubule association (28).
Parallel biochemical reconstitution experiments show that phosphorylation of Hec1 by Aurora B promotes the microtubule dynamics by promoting a weaker Ndc80-microtubule interaction (29). A fundamental characteristic of the kinetochore-spindle microtubule interface is its ability to maintain stable associations while associated microtubules remain dynamic. Such a feature is coordinated by an array of intrinsically low-affinity binding sites which are then modulated by mitotic machinery such as Aurora B kinases. Thus, it would be necessary to generate a specific optical reporter to "read" the spatiotemporal activity of Aurora B quantitatively which will enable us to consolidate HsMis13-Ndc80-microtubule interactions into a model for kinetochore choreography in mitosis. Recent study has indeed demonstrated feasibility of using such a reporter to visualize Aurora B kinase gradient in real-time mitosis (30). It would be of great interest to test reversion of Aurora B phosphorylation dictates the liberation of HsMis13 from kinetochore in real-time mitosis using the aforementioned reporter combined with Aurora B inhibitor.
The aberrant Hec1 and Nuf2 targeting in the HsMis13-depleted cell is likely due to a disruption in a direct HsMis12-Ndc80 interaction given the conserved physical interaction between the Mis12 and Ndc80 complexes. Expression of the phosphomimetic HsMis13 S100/109E mutant in HeLa cells with diminished Aurora B kinase activity restores the kinetochore localization of Hec1 and Nuf2 (~67% of control; Figure  5F) but fails to correct all misaligned chromosomes.
Given the contribution of CENP-H pathway in Ndc80 assembly to the kinetochore (~30%; e.g., ref. 11), it is possible that global inhibition of Aurora B also inhibits the CENP-H pathway which accounts for the defects in chromosome alignment seen in the phosphomimetic HsMis13 S100/109E -expressing HeLa cells.
Taken together, we propose that phospho-regulation of HsMis13 by Aurora B establishes faithful kinetochore-microtubule attachment by recruiting outer kinetochore proteins and correcting aberrant kinetochoremicrotubule attachment errors. It is likely that all of the kinetochore outer plate proteins interact to orchestrate a functional kinetochore during chromosome segregation. The Aurora B-HsMis13 interaction established here is a core of this giant and dynamic complex, which orchestrates kinetochore structure core complex assembly to spindle microtubule attachment in the centromere.

Legends Figure 1. Aurora B interacts with HsMis13 in vitro and in vivo A, co-immunoprecipitation of HsMis13 and GFP-Aurora B from 293T cells. Extracts from cells, transiently
transfected to express GFP-Aurora B (both wild type and kinase-death mutant) and FLAG-HsMis13 or GFP and FLAG-HsMis13, were incubated with antibodies against FLAG (lanes 5-6) and immunoprecipitates (IP) were resolved by SDS-PAGE. Lane 4, immunoprecipitation from GFP-and-FLAG-HsMis13-transfected cell extracts. Western blotting verified co-immunoprecipitation of Aurora B (upper panel; GFP blot) and HsMis13 (lower panel). Note that FLAG-HsMis13 preferentially pulled down wild type but not kinase-death Aurora B. B, Co-distribution of HsMis13 with Aurora B in dividing cells. Asynchronized HeLa cells were stained with HsMis13 rabbit antibody, Aurora B mouse antibody and DAPI. Note that HsMis13 appeared as pairs of separate double-dot from prophase to metaphase. Scale bar represents 10 μm. C, Ser100 and Ser109 of HsMis13 are substrates of Aurora B. Bacterially expressed GST-HsMis13 fusion proteins, both wild type and mutant (S100/109A), were purified and phosphorylated in vitro using [ 32 P]-ATP and active Aurora B as described under "Materials and Methods." Samples were separated by SDS-PAGE. Lower panel Coomassie Brilliant Blue-stained gel of samples of wild type GST-HsMis13 plus Aurora B (GST-HsMis13) and double mutant S100/109A GST-HsMis13 plus Aurora B (GST-HsMis13 S100/109A ). Note that roughly equivalent amounts of GST-HsMis13 protein were present in the two reactions. Upper panel, the same gel was dried and subsequently incubated with x-ray film. Note that there was dramatic incorporation of 32 P into wild type but little into the double mutant, HsMis13 proteins. D, Quantification of 32 P incorporation into HsMis13 protein by Aurora B. The phosphorylation reaction was done as described as Fig. 1C. The 32 P incorporation into HsMis13 proteins was quantified by a PhosphorImager. The 32 P incorporation into wild type HsMis13 and non-phosphorylatable HsMis13 S100/109A was normalized to their protein levels and expressed as a percentage of wild type. Values represent the means ± S.E. of three different experiments. E, HsMis13 is a cognate substrates of Aurora B in vivo. Extracts of nocodazole-synchronized mitotic HeLa cells were immunoprecipitated using an anti-HsMis13 antibody. Immunoprecipitates were then fractionated by SDS-PAGE followed by transferring onto a nitrocellulose membrane. Immunoblotting of HsMis13 immunoprecipitated protein (top panel, Mis13) and serine phosphorylation of HsMis13 (lower pane; Phospho-Mis13). Note that the Aurora B inhibitor VX-680 treatment markedly suppressed HsMis13 phosphorylation but not the protein accumulation. F, Ser100 and Ser109 of HsMis13 are phosphorylated in mitosis. Extracts from mitotic HeLa cells, transiently transfected to express GFP-HsMis13 and GFP-HsMis13 AA (both Ser100 and Ser109 were mutated to Ala), were incubated with an anti-GFP antibody and immunoprecipitates were resolved by SDS-PAGE followed by transferring onto a nitrocellulose membrane. The membrane was first probed for GFP-HsMis13 (upper panel) followed by detection of phospho-serine (lower panel). Lane 1, GFP-HsMis13 immunoprecipitates. Lane 2, GFP-HsMis13 AA immunoprecipitats. Note that GFP-HsMis13 AA was not detected by phospho-serine antibody.   ). B, HsMis13 distributes to the kinetochore at late interphase and departs from kinetochore at the telophase. Asynchronized mitotic HeLa cells were fixed for immunofluorescence staining of HsMis13 (red), ACA (green) and DNA (blue). HsMis13 starts to co-distribute with centromere marker ACA to the kinetochore of late interphase cells (a) and becomes overlapped with ACA staining beginning in prophase (b) and remaining until late anaphase (f). HsMis13 departs from kinetochore in the telophase (g). Scale bar represents 10 μm. C, Phosphorylation of HsMis13 is temporally controlled in mitosis. Aliquots of synchronized HeLa cells were from prophase and telophase as described under "Materials and Methods". Cells were lysed followed by immunoprecipoitation with an anti-HsMis13 antibody. While equal HsMis13 proteins were isolated from prophase and telophase cells (right upper panel), little HsMis13 from telophase cells contains phospho-serine epitopes (right lower panel; lane 2). Probing with an anti-phospho-serine10 antibody confirmed that less abundance of phospho-H3 from telophase cells (left panel; lane 2).

Figure 4. Phosphorylation of HsMis13 by Aurora B is essential for its kinetochore localization A, Expression of exogenous GFP-HsMis13 in HeLa cells.
Samples from both phospho-mimicking and non-phosphorylatable-HsMis13-transfected HeLa cells were prepared and separated on SDS-PAGE, blotted to nitrocellulose, and probed by an anti-HsMis13 antibody. Note that the HsMis13 antibody recognizes both endogenous and exogenously expressed GFP-HsMis13 proteins. B, Exogenously expressed GFP-HsMis13 protein behaves like endogenous HsMis13. HeLa cells were transiently transfected to express GFP-HsMis13 followed by preparation for immunofluorescence staining of HsMis12 (red), GFP-HsMis13 (green) and DNA (blue). GFP-HsMis13 co-distributes with HsMis12 to the kinetochore of mitotic cells (d). Scale bar represents 10 μm. C, Non-phosphorylatable GFP-HsMis13 S100/109A protein failed to localize at the kinetochore. HeLa cells were transiently transfected to express non-phosphorylatable GFP-HsMis13 AA followed by preparation for immunofluorescence staining of HsMis12 (red), GFP-HsMis13 (green) and DNA (blue). GFP-HsMis13 AA failed to distribute with HsMis12 to the kinetochore of mitotic cells (d). Scale bar represents 10 μm. D, Phospho-mimicking GFP-HsMis13 S100/109E protein localizes at the kinetochore. HeLa cells were transiently transfected to express phosphor-mimicking GFP-HsMis13 EE followed by preparation for immunofluorescence staining of HsMis12 (red), GFP-HsMis13 EE (green) and DNA (blue). GFP-HsMis13 EE distributes with HsMis12 to the kinetochore of mitotic cells (d). Scale bar represents 10 μm. Figure 5. Phosphorylation of HsMis13 is essential for faithful assembly of kinetochore A, HsMis13 siRNA suppressed the HsMis13 protein accumulation. Aliquots of HeLa cells were transfected with HsMis13 siRNA and scramble control as described under "Materials and Methods". Cells were then harvested for SDS-PAGE and subsequent western blotting analyses of the efficiency on suppression of HsMis13 protein (upper panel) and specificity of this siRNA-mediated HsMis13 depletion by assessing the protein levels of other kinetochore proteins such as Aurora B, CENP-E, CENP-F, and MAD2 (all lower panels). B, Suppression of HsMis13 prevents assembly of CENP-E but not CENP-F to the kinetochore. HeLa cells were transfected with HsMis13 siRNA oligonucleotide and control oligonucleotide for 48 h followed by fixation and indirect immunofluorescence staining. This set of optical images was collected from HeLa cells stained for HsMis13 (green), ACA (red; c and c'), CENP-E (red; g and g'), CENP-F (red; k and k') and DNA (blue), respectively. As shown in g, CENP-E protein is associated with kinetochore as resolved double-dots in scramble control cells (arrows). However, virtually undetectable staining of CENP-E can be seen in HsMis13-depleted cells (g') while kinetochore-associated CENP-F and ACA signals were not altered by HsMis13 suppression (c' and k'). Scale bar represents 10 μm. C, Quantitation of CENP-E, CENP-F, Nuf2, Hec1 and Aurora B levels at kinetochores of control and siRNA-treated cells. The pixel intensities of HsMis13, Nuf2, CENP-E, CENP-F, Aurora B, and Hec1 (normalized to the ACA signal) in control (closed bar) and HsMis13-repressed (open bars; HsMis13 RNAi) cells were measured. Values represent the means ± S.E. of at least 100 kinetochores in 10 different cells. D, Inhibition of Aurora B prevented kinetochore distribution of GFP-HsMis13. HeLa cells were transiently transfected to express GFP-HsMis13 followed by treatment of VX-680 as described under "Materials and Methods". Cells were then harvested for immunofluorescence staining of GFP-HsMis13 (green), HsMis12 (red) and DNA (blue). Inhibition of Aurora B by VX-680 eliminates the kinetochore localization of HsMis13 (b). Scale bar represents 10 μm. E, Phospho-mimicking GFP-HsMis13 EE restores the kinetochore localization of CENP-E at the kinetochore. HeLa cells were transiently transfected to express phospho-mimicking GFP-HsMis13 EE followed by followed by treatment of VX-680 as described under "Materials and Methods". Cells were then harvested for immunofluorescence staining of GFP-HsMis13 EE (green), CENP-E (red) and DNA (blue). Localization of HsMis13 restores the kinetochore association of CENP-E in the presence of VX-680 (a and b). Scale bar represents 10 μm.  A, immunofluorescence assay of control siRNA-treated cells, Aurora B siRNA-treated cells, GFP-HsMis13 EE -expressing cells and GFP-HsMis13 EE -expressing-and-VX-680-treated cells. B, Quantitation of sister kinetochore distance marked by ACA staining. Kinetochore distance is measured between kinetochores that are marked by ACA staining and localized in the same focal plane as described under "Materials and Methods." Each value from treated samples was calculated from >100 kinetochores selected from at least 10 different cells. DMSO, dimethyl sulfoxide.     Aliquots (0.5 µg per sample) of GST-tagged kinetochore proteins such as Hec1 were dipped onto a nitrocellulose membrane. The membrane was then blocked and incubated with histidine-tagged Aurora B protein (1 µg/ml) for 2 h as described under "Methods". After washing, the blot was then incubated with an anti-Aurora B antibody and developed by using alkaline phosphatase substrates. Note that Aurora B did not react with GST, MBP and BSA, verifying the specificity of HsMis13-Aurora B interaction. Hec1 serves as positive control for this far-western blotting screen.