Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

Funding: This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR MOP-104150 and MOP-111265 to G.J.B.) and the Natural Sciences and Engineering Research Council (NSERC #372593-09 to G.J.B.). M.W. is supported by an NSERC Canada Graduate Scholarship. G.J.B. is the recipient of a CIHR New Investigator Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Centromeres are small structures, so it is easy to see how centromere localization would increase the local concentration of Aurora-B and contribute to kinase activation. In other words, centromeres “cluster” Aurora-B. What remains unclear is how very large structures, such as the microtubules of the mitotic spindle, can accelerate Aurora-B activation. Were Aurora-B to distribute throughout the microtubules of the spindle, the local concentration of Aurora-B may not, in fact, increase, because spreading Aurora-B over such large structures would not lead to clustering. We wanted to understand how microtubules contribute to Aurora-B activation and substrate targeting. Here we show that Aurora-B binds to microtubules and undergoes one-dimensional (1D) diffusion on the lattice. Microtubule binding accelerates Aurora-B activation and increases the phosphorylation of microtubule-associated substrates but decreases the phosphorlyation of substrates that do not bind microtubules. We propose that 1D diffusion on microtubules creates a “reduction in dimensionality” [30] that accelerates Aurora-B kinase activity. This mechanism is similar to the accelerated targeting of site-specific DNA-binding proteins to their sequences [31] .

Enrichment of Aurora-B on the inner centromere is thought to drive activation by increasing the local concentration of Aurora-B [22] . Centromere localization also plays a critical role in current models for sensing proper kinetochore attachments [23] , [24] . Recent work, however, has implicated microtubules in Aurora-B activation; indeed, microtubules are required for the activation of Aurora-B in vitro [25] . We now know that the CPC binds to microtubules via INCENP. The S. cerevisiae homologue Sli5 [26] and human INCENP [27] both co-sediment with microtubules by centrifugation in vitro and co-localize with microtubules in the spindle midzone. The CPC binds to microtubules via INCENP's coiled-coil domain, and deletion of this domain results in a breakdown in spindle assembly in Xenopus egg extracts [28] . This result indicates that active Aurora-B contributes to microtubule polymerization, perhaps through inhibition of MCAK [12] . These interactions create a positive feedback loop, wherein active Aurora-B contributes to microtubule polymerization, which in turn contributes to Aurora-B activation [28] . Microtubule-based activation of Aurora-B was not sufficient for cell-cycle progression in Xenopus extracts, however, as deletion of the centromere-targeting motif of INCENP also lead to failures in spindle assembly [28] . In contrast, Campbell and Desai showed that the centromere localization of the CPC is not essential in budding yeast [29] , suggesting that microtubule localization may be sufficient to enable auto-activation in some organisms. Based on these differing results, the relative importance of centromeres and microtubules in Aurora-B activation is an open question.

Aurora-B becomes active through binding to activators and auto-phosphorylation in trans. Like all protein kinases, Aurora-B has a bilobed structure, including a hydrophobic pocket in the N-lobe that must be filled by a complementary hydrophobic motif in order for the kinase to be active [18] . Unlike other kinases, however, the complementary hydrophobic motif is not provided by C-terminal domains of Aurora-B itself. Rather, the complementary motif is provided by a C-terminal hydophobic region of INCENP, known as the INBox motif [19] . Aurora-B engages in auto-phosphorylation in trans, phosphorylating a crucial threonine on the activation loop that blocks the kinase active site (the T-loop). Human Aurora-B may also dimerize via a domain swap of the activation loop [20] . Further activation is achieved when Aurora-B phosphorylates a site on the INBox (the TSS-motif) [21] . The kinetics of auto-activation depend on the kinase concentration. Aurora-B is constitutively active when purified from E. coli, indicating that a high local concentration of Aurora-B is sufficient to stimulate activation. Similarly, linkage of Xenopus Aurora-B by binding to anti-INCENP antibodies also triggers activation [22] .

The first Aurora kinase, Ipl1, was identified in a screen for genes that cause an increase in ploidy in S. cerevisiae [1] . The family received its name from a Drosophila mutant that formed monopolar spindles reminiscent of the aurora borealis [2] . While fungi have one Aurora kinase (Ipl1 in S. cerevisiae, Ark1 in S. pombe [3] ), metazoans have two kinases that regulate progress through mitosis: Auroras A and B. Aurora-B is part of a protein complex known as the chromosome passenger complex (CPC), so-named because it hitches a ride on chromosomes, which carry the complex to the spindle equator (reviewed in [4] ). The core of the CPC is composed of Aurora-B, INCENP (INner CENtromere Protein, the first component identified [5] ), Survivin [6] , and Borealin (also known as Dasra) [7] . This functional module is conserved from yeast to humans. Alpha-helices of human INCENP, Survivin, and Borealin form a triple-helical bundle that links these CPC components into a single structural unit [8] . After chromosome condensation, the CPC is initially located at the centromere, where Aurora-B helps correct improper kinetochore attachments by phosphorylating several components of the kinetochore [9] , [10] , notably the Ndc80 complex [11] as well as centromeric MCAK [12] , a microtubule depolymerase. Consistent with this, cells enter anaphase with improperly attached and misaligned chromosomes following impairment of Aurora-B function in XTC cells [13] and HeLa cells [14] . At the transition to anaphase, the CPC releases from chromosomes and binds to the spindle midzone. There, Aurora-B creates a gradient of phosphorylation centered on the midzone [15] that functions in spindle midzone organization and cytokinesis [16] , [17] .

Aurora-B is a serine/threonine kinase that controls progression through each phase of mitosis, namely the construction of the mitotic spindle, the segregation of chromosomes, and the completion of cytokinesis. For example, Aurora-B ensures the proper attachment of chromosomes to the microtubules of the mitotic spindle through the correction of microtubules attached to the wrong kinetochore (merotelic attachments). The inability to correct these misattachments is the major source of chromosome instability in cancer cells, which is an underlying cause of aneuploidy. A central question is how Aurora-B activation is governed.

Results

Microtubules restrict Aurora-B activity to MAP substrates The CPC binds to microtubules via the coiled-coil domain of INCENP [28]. In order to reconstitute the interaction of the CPC with microtubules, we expressed and purified a minimal CPC complex consisting of the coiled-coil domain and INBox of INCENP (a.a. 491–873) and Aurora-B, which we have termed CCA (for coiled-coil Aurora-B), using a bicistronic expression vector. The cloning site in the expression vector contained an N-terminal 6 -His tag and a C-terminal EGFP tag followed by a Strep-tag II [32]. The use of two purification tags allowed us to first pull on the INBox via the His tag and then pull on Aurora-B via the Strep-tag II. This purification strategy is designed to remove uncomplexed proteins. Our CCA-GFP purification yielded a highly-purified complex in which Aurora-B and the INBox motif are present in an approximately equimolar ratio (see Figure S1 in File S1). In order to test how microtubules affect the performance of the CCA, we set up in vitro kinase assays with P-ATP. We started the reaction with 1 µM CCA, different concentrations of paclitaxel-stabilized microtubules (from 0 to 6 µM), and 2 µM Histone H3, a conventional Aurora-B substrate that has not been reported to interact with microtubules. After 30 minutes, we measured the intensity of bands in 32P-autoradiograms corresponding to phosphorylated Histone H3 using densitometric analysis. We observed that microtubules reduced the amount of phosphorylated Histone H3. Figure 1A shows a plot of the normalized Histone H3 band intensity at different microtubule concentrations. In the absence of microtubules, we measured a Histone H3 band intensity of = 0.98 0.05 a.u., and this intensity was significantly reduced in the presence of microtubules (e.g., for 3 µM microtubules, = 0.81 0.05 a.u., 0.01, see Fig. 1B for example radiogram). In this experiment, as well as those described below, we also tested a CCA complex with a C-terminal Strep-tag II but without GFP. We observed no differences in the performance of the CCA complex without the GFP tag. There are two interpretations of this result. First, Histone H3 could bind to microtubules in a way that blocks access of Aurora-B to Histone H3. Although Histone H3 has not been reported to bind to microtubules, histones are basic proteins that may interact electrostatically with microtubules, which are acidic. Alternatively, the CCA could interact with microtubules in such a way that the microtubules sequester the kinase activity of Aurora-B away from substrates that do not interact with microtubules. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Microtubules sequester Aurora-B activity toward microtubule-associated substrates. (A) Bar graph showing the normalized Histone H3 band intensity in P radiograms at 3 different microtubule (MT) concentrations. (B) Image of a P radiogram showing radioactive bands corresponding to phosphorylated INBox motif (labeled) and Histone H3 (labeled) at 2 different microtubule concentrations. (C) Bar graph showing the normalized INBox band intensity in P radiograms at 3 different microtubule (MT) concentrations. (D) Image of an SDS-PAGE gel showing bands corresponding to Aurora-B-GFP (labeled), tubulin (labeled), and Histone H3 (labeled) at 2 different microtubule concentrations. The band for the INBox construct, at 52 kDa, is masked by the tubulin band. (E) Bar graph showing the normalized MCAKAAA band intensity in P radiograms at 3 different microtubule (MT) concentrations. (F) Image of a P radiogram showing radioactive bands corresponding to phosphorylated MCAKAAA (labeled) and the INBox motif (labeled) at 2 different microtubule concentrations. https://doi.org/10.1371/journal.pone.0086786.g001 If microtubules sequester Aurora-B, it is possible that the phosphorylation of well-established microtubule-associated substrates will be enhanced. One such substrate is the CCA itself. To assess the effect of microtubles on CCA auto-phosphorylation, we quantified the 32P-autoradiogram bands corresponding to INCENP's INbox motif in the experiment above. Although Aurora-B typically phosphorylates itself in trans prior to purification from E. coli, we observed additional auto-phosphorylation in our in vitro kinase assays. As predicted, the level of CCA auto-phosphorylation increased in the presence of microtubules. Figure 1C shows a plot of the normalized band intensity of the INBox motif at different microtubule concentrations. In the absence of microtubules, we measured an INBox band intensity of = 0.77 0.008 a.u., and this intensity was significantly increased in the presence of microtubules (e.g., for 3 µM microtubules, = 0.98 0.03 a.u., 0.001, see also Fig. 1B). Figure 1D shows an SDS-PAGE gel demonstrating that protein levels remained constant across experiments. These results indicate that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates. In order to determine whether this phenomenon was general or specific to the auto-phosphorylation reaction, we tested a second microtubule-associated substrate, namely the microtubule depolymerase MCAK, a prominent Aurora-B target [12]. Adding MCAK to an in vitro kinase assay with microtubules is tricky, however, because MCAK will depolymerize the microtubules. To prevent MCAK from doing so, we expressed and purified an MCAK construct in which three amino acids essential for depolymerization, the KVD finger, are substituted with alanines (MCAK-KVD AAA, hereafter MCAKAAA [33]). We measured the extent of MCAKAAA phosphorylation by Aurora-B using different concentrations of microtubules, and we observed the microtubules increased the level of MCAKAAA phosphorylation (Fig. 1E). In the absence of microtubules, we measured an MCAKAAA band intensity of = 0.45 0.07 a.u., and this intensity was significantly increased in the presence of microtubules (e.g., for 3 µM microtubules, = 0.7 0.06 a.u., = 0.01, see Fig. 1F for example radiogram). We conclude that microtubules increase the kinase activity of Aurora-B against multiple microtubule-associated substrates, and that microtubules likely play a general role in enhancing Aurora-B activity. We cannot exclude, however, the possibility that the Histone H3 result (Fig. 1A) is explained by the binding of Histone H3 to microtubules in a way that blocks kinase access.

Aurora-B diffuses on microtubules in a 1D random walk We considered two possible mechanisms by which microtubules could enhance the kinase activity of Aurora-B toward microtubule-associated substrates. First, microtubules could induce a conformational change in Aurora-B that enhances its catalytic activity. Alternatively, microtubules could increase the rate at which Aurora-B encounters its substrates. In order to distinguish between these hypotheses, we chose to visualize the interactions of our CCA construct with microtubules using the single molecule assay for microtubule-associated proteins [34]. We expressed and purified a GFP-tagged CCA construct and introduced this protein into a microscope flow chamber with surface-immobilized, rhodamine-labeled microtubules. We observed the CCA binding to the MT lattice and diffusing along it in a one-dimensional random walk. Figure 2A shows a kymograph of this “lattice diffusion,” which is a common mode of interaction for microtubule-associated proteins [35]. We tracked the motion of 500 CCA-GFP molecules using in-house tracking software. Figure 2B shows a plot of the mean-squared displacement of the CCA-GFP against time. We fit the data to a line; the slope of this line gave us a diffusion coefficient of = 0.055 0.001 µm2 s−1. We also measured the lifetimes of the CCA-GFP trajectories (see Figure 2C). The lifetimes were distributed exponentially, with a mean residence time on the lattice of = 1.5 0.02 s, after correcting for photobleaching, from which we can infer a dissociation rate constant of = = 0.66 s−1. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. The CCA diffuses on microtubules via electrostatic interactions. (A) Still image (top) and kymographs (bottom) of the CCA-GFP (green) interacting with microtubules (red). The back-and-forth movements of a 1D random walk are evident. (i) A color-combined kymograph of CCA-GFP diffusion. (ii) An inverted grayscale image of the CCA-GFP signal shown in (i). (B) Plot of the mean squared displacement, against time for the CCA-GFP. A linear relationship is observed, and slope of the fit (blue line), , is related to the diffusion coefficient by . (C) Histogram of the duration of CCA-GFP binding events. An exponential decay fit (blue line) gives a mean duration of = 1.5 s. (D) Plot of the intensity of the CCA-GFP on the microtubule lattice against the concentration of the CCA-GFP. The data is well described by a conventional binding isotherm (blue line). (E) Box plot of the intensity of the CCA-GFP on microtubules at four different salt concentrations. (F) Box plot of the intensity of the CCA-GFP on microtubules for paclitaxel-stabilized microtubules and subtilisin-digested microtubules (labeled). https://doi.org/10.1371/journal.pone.0086786.g002 In order to measure the affinity of the CCA-GFP for microtubules, we measured the fluorescence intensity on the microtubule lattice at increasing CCA-GFP concentrations. Figure 2D shows a plot of the GFP intensity against concentration, which was well-described by a conventional binding isotherm. The fit produced an equilibrium dissociation constant of = 0.08 µM. From the and the , we can infer an association rate constant of = 8.25 µM−1 s−1, which is consistent with the CCA binding to microtubules in a diffusion-limited reaction. These results indicate that the CCA diffuses on the microtubule lattice and that the parameters describing this diffusion are similar to those reported for other microtubule-associated proteins. Most if not all of the microtubule-associated proteins that diffuse on microtubules do so via electostatic interactions with the microtubule lattice, e.g., Kif1A [36], MCAK [37], and the Ndc80 complex [38]. INCENP's coiled-coil domain is enriched in positively charged amino acids and may interact with the negatively-charged C-terminal tails of tubulin [28]. In order to test whether the CCA-GFP diffuses on microtubules by electrostatic interactions, we introduced CCA-GFP molecules to surface-immobilized microtubules in the presence of increasing concentrations of salt. We observed that salt significantly attenuated CCA-GFP binding. Figure 2E shows a plot of the CCA-GFP intensity on microtubules against salt concentration. In the absence of added salt, we measured a CCA-GFP signal of = 277 65 a.u. This signal was reduced by the addition of 25 mM KCl ( = 117 35 a.u., = 50, 0.001), reduced further by the addition of 50 mM KCl ( = 27 15 a.u., = 50), and abolished by further addition of salt. In order to confirm that the CCA-microtubule interactions required the C-terminal tails of tubulin, we removed these tails by proteolytic cleavage with subtilisin. We introduced CCA-GFP molecules into a flow chamber containing both brightly-labeled microtubules and dimly-labeled, subtilisin-digested microtubules. We observed that the GFP signal on subtilisin-digested microtubules were significantly dimmer (see Fig. 2F, = 375 111 a.u. vs. = 89 47 a.u., = 80, 0.001). These results indicate that CCA-microtubule interactions are electrostatic and involve the C-terminal tails of tubulin. Similar results relating to the electrostatic interaction of the CPC with microtubules were recently reported for the budding yeast CPC [39].

Microtubules accelerate Aurora-B auto-activation by a “reduction in dimensionality” In order to understand the role of diffusion in Aurora-B kinase activity, we adopted a simple kinetic model that describes Aurora-B auto-activation. In the model, which is based on the work of Wang and Wu [40] and drawn in Figure 3A, we assume that small amounts of active kinase (labeled ) bind to inactive kinases (labeled ) with an equilibrium dissociation constant, . The active kinase then phosphorylates the inactive kinase with a catalytic rate constant, , producing an additional active kinase, . The Supplemental Information (File S1) contains a derivation of the amount of active kinase that accumulates as a function of time. As a caveat, we note that our model considers kinase activation as a single-step process, when in fact Aurora-B activation is a multi-step process comprised of binding to INCENP and phosphorylation in trans on multiple residues. Nevertheless, a prediction of our model is that decreasing the value of leads to a faster accumulation of active kinase. Our hypothesis is that microtubules change the equilibrium dissociation constant, , by increasing the rate of association of the active and inactive kinases. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Microtubules accelerate Aurora-B activation by a reduction in dimensionality. (A) Schematic of a model for Aurora-B auto-activation. An active kinase ( , bright red) binds to an inactive kinase ( , dim red) with an equilibrium constant, , to form a complex, . The kinase reaction occurs with a catalytic constant, , producing two active kinases (right). (B) Images of P radiograms showing radioactive bands corresponding to phosphorylated INBox motif. The images correspond to 4 microtubule concentrations (labeled at left). The lanes in the radiogram correspond to different time points (labeled at bottom). At higher microtubule concentrations, the radioactive bands become more intense at earlier time points. (C) Plot of the INBox motif band intensity against time for 4 microtubule concentrations. (D) Plot of the mean first passage time, , against the size of the confining space in which the molecules diffuse, . The plot shows both 3D diffusion (red hashed line) and 1D diffusion (green solid line) using the diffusion coefficients for Aurora-B (3D case, estimated; 1D case, measured). 1D diffusion is significantly faster when the molecules diffuse within spaces 2 µm in radius. https://doi.org/10.1371/journal.pone.0086786.g003 We tested the prediction of our model by measuring the amount of active kinase that accumulates over time in the presence of increasing concentrations of microtubules. We began the experiment by treating our purified CCA construct with -phosphatase to partially inactivate the population of kinases, which are otherwise constitutively active due to their expression in E. coli. Next, we inhibited -phosphatase with sodium orthovanadate and started the kinase reaction. The band intensity of the INBox motif on 32P-autoradiograms was measured at six intervals over a 30-minute time course. We observed negligible auto-activation in the absence of microtubules. In contrast, microtubules stimulated auto-activation, and active kinase accumulated faster at higher microtubule concentrations. Figure 3B shows a plot of the INBox motif band intensity as a function of time at 4 different microtubule concentrations. In the absence of microtubules, we observed negligible kinase activity due to the inactivation of the kinase with -phosphatase. At every time point, the level of INBox phosphorylation is higher at increasing microtubule concentration. These results indicate that microtubules accelerate the auto-activation of the CCA complex, which, in our model, implies that microtubules increase the rate of association of active and inactive kinases. How do microtubules increase the rate of association of Aurora-B in trans? One hypothesis is that the diffusion of Aurora-B on microtubules creates a “reduction in dimensionality,” wherein the kinases search for one another by 1D diffusion on the microtubule lattice rather than 3D diffusion is solution [30]. While intuitive, “reduction in dimensionality” is not always effective and depends critically on the ratio of the diffusion coefficients in 3D and in 1D [41]. To test whether “reduction in dimensionality” accelerates the rate of association in our case, we calculated the mean first-passage time for two proteins diffusing in 3D solution, and we compared this to the mean first passage time for two proteins diffusing in 1D on the microtubule lattice. For the 3D case, we used the predicted diffusion coefficient of a globular protein of 100 kDa, whereas for the 1D case, we used the diffusion coefficient for the CCA-GFP on microtubules measured above. Our model assumes the proteins are confined within a space of known size (e.g., within a cell). Figure 3D shows a plot of the mean first passage times as a function of the size of the confining space. Our model shows that, for molecules confined within spaces of 2 µm in radius, 1D diffusion confers a 6-fold advantage in mean first passage time, an advantage that grows exponentially as the confining space becomes larger. The mean first passage time may be further reduced if the CCA complexes undergo interspersed periods of 3D and 1D diffusion, as occurs in most models for the interaction of site-specific DNA-binding proteins with their target sequences [31].