The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes.


Introduction
Executive decisions concerning when cells enter and exit mitosis are made by Cdk1 with cyclins A and B as cofactors. Once cells have entered mitosis, Plk1 and the Aurora kinases direct spindle formation, regulate chromosome attachments to spindle microtubules, ensure the operation of the spindle checkpoint, and enable daughter cells to complete cytokinesis (reviewed in [1][2][3][4]). Plk1 and Aurora A also function in the regulation of mitotic entry (reviewed in [5]).
In higher eukaryotes, Plk1 and Aurora B have potentially antagonistic activities during the early stages of chromosome attachment and alignment on the mitotic spindle. Plk1 phosphorylation of kinetochore components and microtubule plus-endassociated proteins is required for the establishment of stable kinetochore-microtubule (KT-MT) interactions. Electron micrographs of human cells treated with the Plk1 inhibitor BI2536 show fewer microtubule connections per kinetochore [6]. Tensionsensitive phosphorylation of BubR1 by Plk1 regulates the initial stability of KT-MT interactions [7], as do phosphorylation of CLIP-170 [8] and NudC [9]. Plk1 also phosphorylates components of the Ska and KNL-1/Mis12/Ndc80 (KMN) kinetochore complexes as well as centromere proteins CENP-B, CENP-C, CENP-E, and CENP-F. However, the function of these phosphorylations is not known [10].
The chromosomal passenger complex (CPC), consisting of Aurora B kinase, INCENP, Survivin, and Borealin [11], has a role in the correction of kinetochore-microtubule attachment errors by promoting the release of kinetochore-microtubule attachments [11][12][13][14][15]. The localization of the CPC relative to kinetochores is critical for regulation of kinetochore-microtubule attachments [16]. CPC targeting to inner centromeres occurs as a result of Survivin binding to histone H3 phosphorylated on Thr3 by Haspin kinase [17][18][19] and is helped by an Aurora B-dependent positive feedback loop [20].
The temporal and spatial regulation of Plk1 activation is complex. While Polo activity is not required for mitotic entry in unperturbed cell cycles, its activation by T-loop phosphorylation is needed for its functions during mitosis. At centrosomes, Aurora A kinase-Bora phosphorylates Plk1 on Thr210, thereby activating Plk1 at the G2-M transition in human cells [21,22]. Subsequently, Plk1 triggers the degradation of both Bora [23,24] and Aurora A [25]. The regulation of Plk1 activity at kinetochores is a critical and largely unstudied question.
Here, we have examined the mechanism of Polo kinase activation at Drosophila and human kinetochores. We show that Aurora B is required for Polo T-loop phosphorylation at the centromere, but not at centrosomes. Our studies identify a new regulatory link between the Aurora B and Polo kinases mediated by INCENP. Furthermore, we demonstrate that this mechanism of regulation of Polo kinase at the kinetochore by the CPC is conserved in human cells. These results support our previous hypothesis that INCENP acts as a platform coordinating the activities of these kinases on chromosomes during early mitosis [26].

Results
Polo Kinase Localizes to the Centromere/Kinetochore Region Before the CPC Does As a starting point to examine the relationship between Polo and the CPC in Drosophila, we compared their localization in space and time. INCENP and the CPC are diffuse during early prophase in Drosophila cultured cells ( Figure 1A2). In contrast, Polo kinase in these early stages is localized at centromeres (Figure 1, arrows; Figure S1A), at centrosomes, where it colocalizes with Aurora A ( Figure 1A-D, asterisks; Figure S2), and also at the nuclear envelope ( Figure 1A3, blue arrowhead), where it has been proposed to promote nuclear envelope breakdown (NEB) [8].
Later during prophase and early in prometaphase, INCENP concentrates at specific, brightly stained chromosomal regions that probably correspond to heterochromatin. At this time, Polo kinase is already concentrated at centromeres ( Figure 1B3). This is the earliest stage at which we observe partial colocalization between Polo ( Figure 1B3,4 white arrows) and the CPC.
In metaphase, when chromosomes are bioriented and under tension, INCENP is concentrated on inner centromeric threads that extend between bioriented sister kinetochores, running parallel to microtubules (Figure 1C2,4; Figure S1D). At this stage, most chromosome-associated Polo is detected in the outer kinetochore ( Figure 1C3,4, arrows) and does not colocalize with INCENP. Later, in early anaphase, INCENP and Polo are observed on threads parallel to central spindle microtubules ( Figure 1D and unpublished data), although prominent Polo labelling is still detected at the kinetochore.
We conclude that Polo concentrates at centromeres before INCENP does and that both proteins transiently colocalize there during early prometaphase.

INCENP and Polo Interact in vitro and in vivo at the Centromere in Early Mitosis
To ask if this colocalisation of INCENP and Polo reflects a direct interaction between the two proteins, soluble, bacterially expressed, full-length Drosophila GST-INCENP ( Figure 1E right panel) was mixed with in vitro-translated Polo kinase using Aurora B and luciferase as positive and negative controls, respectively. The mixture was then incubated with glutathione beads and bound proteins detected by SDS-PAGE. Robust binding was observed between GST-INCENP and Polo or Aurora B, but not with the luciferase control ( Figure 1E). Interestingly, this interaction did not require CDK phosphorylation of INCENP, as previously described for the binding of mammalian INCENP to Plk1 [27].
We confirmed that a physical interaction between INCENP and Polo also occurs in vivo by immunoprecipitation from cell extracts. Cell lines stably expressing either Polo-GFP or Aurora B-GFP (positive control) were lysed and the tagged protein immunoprecipitated with anti-GFP. INCENP was readily detected in both immunoprecipitates by immunoblotting ( Figure 1F).
To determine more precisely when and where INCENP and Polo interact during the cell cycle, we used a proximity ligation assay (PLA) to map sites where INCENP, Polo, and Aurora B are in close proximity. PLA is based on conventional double staining using primary antibodies raised in different species. The secondary antibodies used for detection are tagged with short DNA oligonucleotides. If those oligonucleotides are close enough to allow them to be bridged by hybridization with circle-forming oligonucleotides (distance between antigens of 10-30 nm, [28]), the circle can be amplified by rolling circle DNA synthesis and a positive PLA signal is obtained. That signal requires not only the close proximity of the antigens but also their favourable spatial conformation and absence of structural obstacles so that the oligos can interact and subsequent reactions take place. Thus, only a subset of actual interactions between proteins is detected with the PLA technique.
We validated the PLA assay by first confirming the known interaction between endogenous INCENP and Aurora B [29][30][31][32]. Indeed, we readily observed PLA signals associated with chromosomes in prophase and prometaphase cells ( Figure 1G1).

Author Summary
When cells divide, their chromosomes segregate to the two daughter cells on the mitotic spindle, a dynamic macromolecular scaffold composed of microtubules. Each chromosome consists of two sister chromatids. Microtubules attach to the chromatids at structures called kinetochores, which assemble at the surface of the constricted centromere region where the sister chromatids are most closely paired. To segregate correctly, sister kinetochores must attach to microtubules emanating from opposite spindle poles. Kinetochore attachment to microtubules occurs randomly and mistakes occur frequently. For example, both sister kinetochores may attach to one pole, or one kinetochore may attach to both poles simultaneously. Two protein kinases, Aurora B and Polo, have essential roles in regulating this process: Aurora B triggers the release of incorrect attachments and Polo strengthens the grip that correctly attached kinetochores have on microtubules. In this work, we have investigated the potential functional links between these two crucial enzymes at centromeres in cells of the fruitfly. We found that early in division, Aurora B and Polo both interact with a structural partner protein named INCENP at centromeres. This allows Aurora B to phosphorylate Polo, thereby activating it. We show that coordinating the activities of these two central mitotic kinases is crucial for successful cell division, and that this mechanism is conserved in human cells.
This confirmed a recent study that found positive PLA signals between various members of the CPC in all phases of mitosis in human cells [33].
In Drosophila, the positive PLA signals overlapped with Aurora B-GFP, confirming that epitopes on INCENP and Aurora B are in close proximity in the inner centromere ( Figure 1G4). A parallel assay using antibodies against INCENP and GFP confirmed this close association of INCENP with exogenous GFP-tagged Aurora B (unpublished data). In two negative controls, we failed to observe PLA signals using antibodies to INCENP and c-tubulin and between Polo and the centromere histone CENP-A/CID ( Figure 1J,K).
We next used PLA to define where in cells interactions occur between INCENP and Polo. Cells co-stained for INCENP and Polo showed PLA signals on chromosomes in early mitosis ( Figure 1H1). These signals colocalized with Aurora B-GFP, confirming that the interaction occurs at inner centromeres ( Figure 1H4). PLA also detected a close association between INCENP and Polo T182ph , the activated form of Polo kinase, using an anti-phospho-epitope-specific antibody ( Figure 1I1, see validation of the antibody below). These positive PLA signals were also present in inner centromeres (Figures 1I4).
We conclude that INCENP and Polo physically interact and are in close proximity at inner centromeres during early mitosis in Drosophila.

INCENP Is Required for Polo Kinase Activation at the Inner Centromere
The association between INCENP and Polo described above suggested that INCENP and other CPC components might have a role in Polo activation by T-loop phosphorylation. To test this hypothesis, we asked whether reducing INCENP protein levels by RNAi affected the localization and activation of Polo kinase, as detected by monitoring Polo T-loop phosphorylation. Plk1 phosphorylation on the highly conserved T-loop residue Thr-210 (Thr-182 in Drosophila Polo, Figure 2A) is crucial for kinase activation [34]. T182 of Drosophila Polo is a major phosphorylation site detected by mass spectrometry, and a phosphomimetic mutation at that site (Polo T182D ) increases Polo kinase activity in vitro (VA, unpublished observations).
To examine Polo activation, we used a phospho-epitope-specific antibody raised against human Plk1 T210ph that also recognises Drosophila Polo T182ph ( Figure 2B). In asynchronous cultures, Polo T182ph was barely detectable by Western blotting. However, we could readily observe endogenous Polo T182ph using this antibody after treatment with the phosphatase inhibitor okadaic acid (OA; Figure 2B). This signal disappeared following Polo depletion by RNAi, confirming that it comes from the Polo protein (see below). Moreover, Polo-Myc gives a signal at the expected higher molecular weight, while Polo T182A -Myc and Polo T182D -Myc are not recognized by this antibody ( Figure 2B). In these experiments, Polo was detected as a doublet by Western blotting. This mobility shift is not caused by T182 phosphorylation and may be caused by an as-yet uncharacterized modification.
We used this antibody to examine the distribution of Polo T182ph by immunofluorescence in cycling DMel-2 cells that had not been treated with okadaic acid. The antibody detected Polo T182ph at centromeres/kinetochores, centrosomes, the cleavage furrow, and midbody. This staining was largely abolished following Polo RNAi-depletion ( Figure S3).
Although most Polo accumulates at prometaphase kinetochores ( Figure 2C3; Figure  . This is consistent with the observation that Polo localization to this region precedes that of INCENP ( Figure 1A). Importantly, we could still readily detect active Polo T182ph at centrosomes of cells following INCENP knockdown ( Figure 2E4,F4, asterisks).
These experiments reveal that INCENP is required for T182 phosphorylation and activation of Polo kinase at inner centromeres in early mitosis. Activation of centrosomal Polo does not require INCENP.

Polo T-Loop Phosphorylation Is Required for Mitotic Progression But Not For Mitotic Entry
In order to investigate the function(s) of Polo T182 phosphorylation in mitosis, we established stable cell lines allowing inducible expression of Polo WT -GFP or Polo T182A -GFP. Endogenous Polo could be depleted in those cells by RNAi against the 39UTR of the native transcript ( Figure 3A). Expression of Polo WT -GFP rescued the viability and proliferation of cells depleted of endogenous Polo. However, expression of Polo T182A -GFP did not, and cells died (unpublished data). Thus, Polo T-loop phosphorylation is essential for viability.
Polo-depleted cells accumulated in mitosis, exhibiting phenotypes similar to those observed for the first polo mutants [35]. Expression of Polo WT -GFP restored mitotic progression in cells depleted of endogenous Polo ( Figure 3B), but expression of Polo T182A -GFP did not. Cells expressing only Polo T182A -GFP accumulated in prometaphase/metaphase ( Figure 3C), often with  The phosphoespecific antibody anti-Plk1 T210Ph recognises Drosophila Polo T182Ph , both Myc-tagged and endogenous (e). Both endogenous and Myctagged Polo T182Ph were detected as a doublet, suggesting that they can be modified at another site. The asterisk indicates a non-specific band that does not disappear after Polo RNAi. This band increases following okadaic treatment, and therefore could correspond to a non-specific phosphoepitope. unaligned chromosomes ( Figure 3D-F). Interestingly, while the loss of Polo led to an increase in monopolar spindles, substitution of endogenous Polo with Polo T182A -GFP did not ( Figure 3D-F). This suggests that T-loop phosphorylation of Polo may be dispensable for its role in bipolar spindle assembly.
The observation that INCENP-dependent activation of Polo by phosphorylation at T182 at centromeres/kinetochores is required for chromosome alignment in prometaphase is consistent with the known role of Polo in regulating kinetochore function.

Aurora B Activity Is Required for Polo Kinase Activation at Centromeres
Because the best known role of INCENP is to activate Aurora B kinase in the CPC, we next asked whether Aurora B has a role in Polo T-loop phosphorylation at centromeres. Drosophila Polo T182 (corresponding to human Plk1 T210) is preceded by a conserved stretch of basic residues resembling the consensus site for Aurora kinases (Figure 2A) [31,36,37]. Indeed, Drosophila Aurora B complexed with a fragment of INCENP can directly phosphorylate Polo in vitro ( Figure 4A). A T182A mutation in the Polo used as a substrate reproducibly reduced its phosphorylation by about one half. Thus, Polo T182 is a major phosphorylation site for Aurora B ( Figure 4A). Similar results were obtained using human Aurora B on GST-Polo WT or GST-Polo T182D (unpublished data).
Kinase inhibition studies suggest that Aurora B is responsible for Polo T182 phosphorylation in vivo. Binucleine 2 is the only specific Aurora B kinase inhibitor described to date that is effective in Drosophila cells [38,39]. When DMel-2 cells were treated with 20 mM Binucleine 2 for 2 h, H3 S10ph was undetectable in mitotic cells (unpublished data; [39]) and INCENP and Aurora B were dispersed in clumps on the chromosomes ( [38,39]; Figure S5B,C, compare with Figure S5A). Both of these phenotypes are characteristic of the loss of Aurora B function [40,41].
Aurora B kinase activity is required for Polo activation at kinetochores, and levels of kinetochore-associated Polo T182ph were greatly reduced in Binucleine 2-treated mitotic cells (Figure 4B4-D4; Figure 4E). In contrast, we observed no obvious difference in the localization of bulk Polo kinase in those cells (Figure 4B3-D3; Figure 4E). Importantly, as in the case of INCENP RNAi, we could still detect activated Polo kinase at centrosomes in the same cells ( Figure 4B4-D4 asterisks).
As independent confirmation of the inhibitor studies, RNAimediated depletion of Aurora B also led to disappearance of the Polo T182ph signal observed in Western blots after OA treatment of cells, while total Polo levels remained unchanged ( Figure 4F). In striking contrast, the Polo T182ph signal actually increased after partial Aurora A depletion (Figure 4Fa), perhaps because cells accumulated in mitosis.
The above results suggested that Aurora B rather than Aurora A plays a major role to promote Polo T182 phosphorylation at centromeres in Drosophila cells. In order to exclude that our failure to detect Polo T182ph by Western blotting following Aurora B depletion was due to a cell cycle block outside mitosis caused by OA treatment, we examined the effects of RNAi depletion of Aurora A, Aurora B, and INCENP on the Polo T182ph signal at centromeres in individual mitotic cells without okadaic acid treatment. Brief (3 d) dsRNA treatments were used to avoid an accumulation of binucleate cells caused by failure in CPC function in cytokinesis.
Depletion of Aurora B or INCENP led to a significant reduction of the Polo T182ph signal at centromeres ( Figure 4G). This effect was specific to centromeres, and Polo T182ph levels at centrosomes were unaffected following depletion of Aurora B or INCENP ( Figure  S6A). In contrast, Aurora A depletion had no effect on levels of Polo T182ph at centromeres, but led to a modest reduction in Polo T182ph levels at centrosomes.
Together, these results confirm that Aurora B and INCENP are required for Polo activation at the centromere/kinetochore in early mitosis and strongly implicate Aurora B as the kinase responsible.

The CPC Is Required for Polo Activation at Kinetochores in Larval Neuroblasts
The CPC is required for Polo kinase activation at centromeres in live animals, and not only in aneuploid cultured cells. To demonstrate this, we examined flies homozygous for the hypomorphic female-sterile allele incenp QA26 , a point mutation in the highly conserved IN-box domain [42].
We observed a strong signal of Polo T182ph concentrated at kinetochores in wild-type mitotic neuroblasts ( Figure 5A3). In third instar larval neuroblasts from the incenp QA26 mutant, 27% of mitoses (n = 290) showed obvious defects in INCENP localization, with the protein spreading onto chromosome arms ( Figures 5B2,  S5E). This was never observed in wild-type neuroblasts (n = 303; Figures 5A, S5D). The incenp QA26 mitotic phenotype ( Figure S5E Figure 5B3; Figure 5E). In contrast, overall levels of Polo at kinetochores remained similar to wild type ( Figure 5G and I).
To test if Polo T182 phosphorylation requires Aurora B activity in vivo, we dissected whole brains, treated them with Binucleine 2, and processed them for immunostaining as above. After a 2-h incubation in 20 mM Binucleine 2, Histone H3 S10ph (a reporter for Aurora B activity) was undetectable in mitotic cells (unpublished data). Drug-treated neuroblasts also showed the characteristic dispersion of INCENP in clumps on chromosome arms ( Figure 5C2,D2 arrowheads; Figure 5H).
As predicted, Polo T182ph was virtually undetectable at kinetochores of Binucleine 2-treated neuroblasts, but remained readily observable at centrosomes ( Figure 5C3,D3 asterisks). Total Polo levels at kinetochores remained similar to wild type in these cells. Thus, both Aurora B activity and INCENP are required for Polo  As independent confirmation that the CPC contributes to Polo regulation in vivo, we conducted a genetic experiment in a sensitized background. Tubulin-Gal4-driven overexpression of Polo mutated in a conserved destruction box (PoloDdb- Figure  S7) is semi-lethal at the pupal stage, suggesting that excessive Polo activity is detrimental to development. Interestingly, a decrease in the levels of functional INCENP rescued the semi-lethality of PoloDdb-expressing flies. This was observed when one wild-type allele of incenp was replaced with either of the alleles incenp EC3747 or incenp QA26 ( Figure S7). A heterozygous deletion removing Aurora B did not cause a significant rescue in the same assay, suggesting that INCENP may be the limiting CPC component for Polo regulation.
Together, these results confirm that the CPC contributes to the regulation of Polo function at kinetochores in vivo.

The CPC Is Required for Plk1 Activation at Kinetochores in Human Cells
Importantly, the regulation of Polo T-loop phosphorylation described above for Drosophila is conserved in human cells. We used siRNAs to deplete either Aurora B ( Figure 6A) or INCENP ( Figure 6C) in HeLa cells and subsequently measured the levels of total Plk1 and Plk1 T210Ph at kinetochores. Both INCENP and Aurora B depletion caused a dramatic reduction in Plk1 T210Ph levels in early mitosis (Figure 6Ad3,B,Cd3,D). Levels of Plk1 were also slightly reduced, confirming that the CPC is at least partly required for the stable localization of Plk1 to mammalian kinetochores [27]. Comparable results were obtained when we treated cells with the Aurora B kinase inhibitor ZM447439 ( Figure  S8), confirming that Aurora B activity is indeed required for the presence of Plk1 T210Ph at kinetochores in human cells.
Our results thus indicate that the CPC activates Polo kinase by T-loop phosphorylation at centromeres in both flies and humans.

Discussion
Coordination of Polo and Aurora B activity at kinetochores is critical in early mitosis, as the two kinases play potentially antagonistic but complementary roles in regulating kinetochoremicrotubule interactions. Aurora B is essential for the correction of aberrant attachments [13][14][15], and indeed, tethering Aurora B too close to kinetochores interferes with the formation of stable attachments [16]. In contrast, Plk1 activity is required for initial stabilisation of microtubule attachments to kinetochores [7][8][9]. We suggest that interactions with INCENP may provide a mechanism to coordinate the activities of these two essential kinases during early mitosis.
Recent studies suggest that Plk1 is activated at centrosomes when its T-loop (T210) is phosphorylated by Aurora A kinase-Bora, and that this promotes the G2/M transition upstream of Cdk1 [21,22], although Polo activity is not required for mitotic entry ( [3]; this paper- Figure 3). How Plk1 is activated at kinetochores remained an important unsolved question. Our present results show that Aurora B and INCENP, which are concentrated at inner centromeres [43,44], function there to activate Polo by phosphorylating its T-loop.
Plk1 recruitment to centromeres in late G2 has been variously proposed to be mediated by Bub1 [45], INCENP [27], and BubR1 [7]. Another report implicated the self-primed interaction of Plk1 with PBIP1/CENP-U [46]. This could potentially explain why Plk1 activity is reportedly required for its localisation to kinetochores in human cells [47].
Our RNAi studies confirmed that Plk1 is partially dependent on the CPC for its centromeric localization in human cells. However, this appears not to be the case in Drosophila, where Polo is present at centromeres before NEB, at a time when INCENP is not yet concentrated at inner centromeres and before Polo T182ph , the active form of the kinase, is detected there. Indeed, we observed no significant decrease in kinetochore-associated Polo levels after INCENP RNAi in Drosophila cells.
Although Polo targeting to kinetochores is independent of the CPC in Drosophila, its activation there does require the CPC with active Aurora B. Our data suggest that INCENP binding to Polo facilitates its subsequent activation by Aurora B kinase ( Figure 7B,C). Indeed, INCENP and Polo interact physically in vitro and co-immunoprecipitate in mitotic cell extracts. Although most centromeric Polo kinase is concentrated in the outer kinetochore in prophase and prometaphase, active Polo (Polo T182ph ) is also found in inner centromeres, where it overlaps with INCENP as confirmed by a proximity ligation assay (PLA).
A range of evidence presented here suggests that Aurora B is the upstream kinase responsible for Polo kinase activation at centromeres. Firstly, Aurora B phosphorylates Polo at Thr182 in vitro. Secondly, RNAi depletion of INCENP or Aurora B, but not Aurora A, reduces levels of active Polo T182ph at kinetochores. Thirdly, tissue culture cells and third larval instar neuroblasts treated with a specific inhibitor of Drosophila Aurora B kinase show decreased levels of Polo T182ph at kinetochores. In all of the preceding experiments, Polo T182ph levels are affected at kinetochores but not at centrosomes, where Polo is presumably activated by Aurora A [21,22]. Importantly, this involvement of Aurora B in Polo activation at centromeres discovered in Drosophila is conserved for Plk1 in human cells.
Our results suggest a model for interactions between Polo kinase and the CPC at centromeres (Figure 7). In Drosophila cells, Polo targets to centromeres before the CPC is recruited by Survivin binding to histone H3T3ph [17][18][19]. At inner centromeres of chromosomes whose kinetochores are not under tension, Polo now binds to INCENP. This promotes Polo kinase activation, as Aurora B phosphorylates Polo T182 . We suggest that interactions with INCENP link the two complementary kinase activities, thereby potentially creating a microtubule attachment/detach-kinetochore pairs showing decreased levels of Polo T182Ph . (E) Dot plot showing the quantification of INCENP/Polo/Polo T182Ph signal intensity at the kinetochore (t test: *** p,0.0001; n.s., not significant; p = 0.4028). Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data was plotted using KaleidaGraph software. (F) RNAi depletion of Aurora B, but not Aurora A, strongly reduces Polo T182Ph levels in DMel-2 cells treated with okadaic acid. Cells were transfected with the indicated dsRNAs for 4 d, and 100 nM okadaic acid added for 4 h before immunoblotting to improve visualization of phosphorylated Polo. A dsRNA against the Kanamycin resistance bacterial gene was used as a negative control. Asterisks: non-specific bands. Both bulk Polo and PoloT 182ph appear as doublets. (G) RNAi depletion of Aurora B or INCENP, but not Aurora A, reduces Polo T182Ph levels at centromeres/kinetochores. Cycling cells were treated with the indicated dsRNAs for 3 d (immunoblots are shown in Figure S6B) and Polo T182Ph was detected by immunofluorescence. Levels of Polo T182Ph at centromeres/kinetochores in prometaphase and metaphase cells were measured at individual kinetochores using Image J, subtracting background (Kt-bkd). Asterisks indicate centrosomes. Error bars = S.E.M. doi:10.1371/journal.pbio.1001250.g004 Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data was plotted using KaleidaGraph software. (F-I) Levels of Polo kinase are not affected ment cycle at kinetochores. Such a cycle would not be possible without a balancing phosphatase activity, and PP2A-B56 has recently been shown to oppose both Aurora B and Plk1 activities at kinetochores to promote stable attachments [48].
At metaphase, when chromosomes are bioriented and under tension, the CPC and Polo kinase exhibit only a partial overlap. A weakening of the INCENP/Polo PLA signals in metaphase suggests that Polo may be released from INCENP after its activationpossibly moving to the outer kinetochore ( Figure 7D). During metaphase, the CPC localizes in the inner centromere, stretching between sister kinetochores, whereas Polo and Polo T182ph concentrate mainly at the kinetochores. This separation may be necessary to allow Polo-mediated stabilisation of kinetochore-microtubule attachments. The coordinated activities of both kinases at kinetochores and their tension-mediated separation might facilitate a dynamic equilibrium between attached and unattached kinetochores, selectively stabilizing proper chromosome attachments.
In summary, our results reveal that INCENP and Aurora B are responsible for Polo kinase activation at centromeres but not at centrosomes during mitosis. These experiments support the hypothesis that INCENP acts as a scaffold integrating the crosstalk between these two important mitotic kinases [26].

Drosophila Strains
Fly strains were grown at 25uC in standard Drosophila medium.
Cells were treated with either DMSO or 20 mM Binucleine-2 for 2 to 4 h before being processed for immunostaining as described previously [41]. For experiments shown in Figures 3,  4F,G, and S6, 1.2610 6 D-Mel2 cells were transfected in 6-well plates with 20 mg of dsRNA using Transfast reagent (Promega). Cells were analysed 3 or 4 d later by immunofluorescence and immunoblotting. The control dsRNA was generated against the sequence of the bacterial Kanamycin resistance gene. For experiments shown in Figures 3, 4G, and S6A, cells were seeded on coverslips and treated for 10 s in BRB-80+0.1% NP-40 before a 20 min fixation in BRB-80+4% formaldehyde. Cells were then permeabilized for 10 min in BRB-80+0.1% Triton X-100 and blocked for 1 h in PBS+0.1% Tween20+1% BSA. Primary antibodies were diluted in PBS+0.1% Tween20+1% BSA and incubated overnight at 4uC. Secondary antibodies were incubated 2 h at room temperature. Coverslips were mounted with Vectashield+DAPI. Images were taken using an AxioImager epifluorescence microscope.

Proximity Ligation Assay (PLA)
Proximity Ligation Assay was performed using Duolink QL (Olink, Uppsala, Sweden) following the manufacturer's protocol. Duolink anti-rabbit plus probe, anti-mouse minus probe, and antirat minus probe were used. The following antibody pairs were used for the assay: Rabbit polyclonal anti-Incenp Rb801 [41] For each antibody pair, exponentially growing DMel-2 cells were seeded on Con-A treated coverslips and fixed for immunostaining as described previously [41]. After overnight incubation with primary antibody at 4uC, half of the samples were processed following the normal immunostaining protocol [41] and the other half was used for the PLA assay.
Imaging was performed using Olympus IX-71 microscope controlled by Delta Vision SoftWorx (Applied Precision, Issequa, WA, USA). Image stacks were deconvolved, quick-projected, and saved as tiff images to be processed using Adobe Photoshop. Linescans were generated using Image-Pro software.

Kinase Assay
HIS-Polo and HIS-Polo-T182A were expressed in BL21 bacteria from pETDuet-based constructions (see above). Protein purification was done using Talon resin (Clontech) and purified proteins were stored on the resin at 280uC.
For the kinase assay, HIS-Polo and HIS-Polo-T182A on the resin were incubated with Drosophila Aurora B in complex with INCENP 645-755 [39] for 15 min at 30uC in 20 mM K-HEPES pH 7.5, 2 mM MgCl 2 , 1 mM DTT, 500 mM ATP, 5 mCi 32 P-g-ATP. Reactions were initiated by the combination of the substrate-bound resins to a fixed volume of a master mix containing all other reagents. Reactions were stopped by the addition of Laemmli sample buffer. Samples were separated by SDS-PAGE and transferred to nitrocellulose. Quantitative, subsaturation measurements of radioactivity and Polo Western blot signals were obtained using a PhosphorImager and a Typhoon luminescence reader, respectively.
RNAi experiments were performed using annealed siRNA oligos (Qiagen) diluted in serum free OptiMem and transfected  using HiPerFect reagent (Qiagen) according to the manufacturer's protocol. HeLa cells were seeded on coverslips at a density of 1610 5 cells/ml and diluted siRNA was added to cells so that the final concentration of siRNA was 40 nM. Coverslips were fixed at 48 h. For control transfections non-silencing random scramble siRNA oligos were used at the same concentration. The full sequences of siRNA oligos used are as follows: for Aurora B siRNA, 59-AACGCGGCACTTCACAATTGA-39; for INCENP siRNA 59-AGATCAACCCAGATAACTA-39. For drug treatments, ZM447439 (Tocris Bioscience) or DMSO as control were added to the cells at the concentration of 3 mM for 1 h.
Quantification of Plk1 and Plk1 T210Ph on approximately 1,000 centromeres per condition was carried out as follows: Deconvolved images were imported into OMERO [54] and segmentation of centromere foci (ACA, Cy5, reference channel) performed using Otsu segmentation implemented in Matlab. Masks stored in OMERO were then used to calculate intensities, and output into comma-separated value file for plotting in Excel. or not for 60 h, fixed, and stained for pT182-Polo and alphatubulin or CENP-A (centromere). The pT182-Polo stainings at centromeres/KTs and centrosomes are largely abolished. pT182-Polo stainings of the centrosomes and the midbody in cytokinesis were strongly diminished, but never completely abolished, probably because cells that could complete mitosis were those for which Polo depletion was only partial. In addition, we always observed a non-specific staining of unknown nature at or near the DNA, which remained visible during mitosis in Polo-depleted cells more than in control cells. (TIF) Tubulin-Gal4 driver were identified by the absence of phenotypic markers from balancer chromosomes. The number of flies obtained relative to the expected numbers (one-fourth of the total progeny) is shown for each incenp genotype. N, total numbers of progeny obtained and scored from three vials for each cross. The only definitely null aberrations that we could obtain were large deficiencies that could not be tested because they interacted with balancer chromosomes in our crosses; therefore, it remains formally possible, although unlikely, that both incenp alleles tested here have antimorphic effects. (TIF) Figure S8 Aurora B activity is required for activation of Plk1 at centromere/kinetochores in human cells. (A) HeLa cells treated with DMSO or ZM447439 immunostained for Histone H3 P-Ser10 (green), Plk1 (red), or Plk1 T210Ph (red) and DNA (blue). Scale bar = 10 mm.