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During vertebrate mitosis, the centromere-associated kinesin CENP-E transports misaligned chromosomes to the plus ends of spindle microtubules. Subsequently, the kinetochores that form at the centromeres establish stable associations with microtubule ends, which assemble and disassemble dynamically. Here we provide evidence that after chromosomes have congressed and bi-oriented, the CENP-E motor continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends. Using a combination of single molecule approaches and laser trapping in vitro we demonstrate that once reaching microtubule ends, CENP-E converts from a lateral transporter into a microtubule tip-tracker which maintains association with both assembling and disassembling microtubule tips. Computational modeling of this behavior supports our proposal that CENP-E tip-tracks bi-directionally via a “tethered motor” mechanism, which relies on both the motor and tail domains of CENP-E. Our results provide a molecular framework for CENP-E's contribution to the stability of attachments between kinetochores and dynamic microtubule ends.
Accurate chromosome segregation depends on interactions between microtubules (MTs) and kinetochore, a protein structure localized at each centromere1. Initially, kinetochores often attach to the walls of MTs with the chromosomes then moving toward a spindle pole in a dynein-dependent manner2,3. The pole-proximal chromosomes then need to congress, i.e. move to the spindle midzone where MT plus ends are located. The centromere protein CENP-E, a MT-dependent plus-end directed motor of the kinesin-7 subfamily, is essential for congression of initially misaligned chromosomes4–8. Perturbation of CENP-E function using antibody injection9 or depletion10, gene inactivation11, siRNA depletion12, or inhibition with a small molecule inhibitor13 blocks MT plus-end-directed motion of pole-proximal chromosomes, accompanied by a failure of both kinetochores to form stable MT attachments11.
Previous work has suggested that the role of CENP-E in mitosis is not limited to the transport of chromosomes during congression. First, CENP-E is detected at the kinetochores of already congressed chromosomes14,15. Second, depletion of CENP-E induces up to a 50% reduction in the number of MTs on congressed chromosomes11,16. Third, CENP-E gene deletion leads to an increased proportion of lagging chromosomes in anaphase in mouse liver cells and embryonic fibroblasts11,17. Fourth, after CENP-E-mediated congression, CENP-E-dependent localization of protein phosphatase 1 (PP1) to kinetochores is still required for stable microtubule capture by those kinetochores8. Furthermore, antibodies to CENP-E block the motions of isolated mammalian chromosomes at the ends of depolymerizing MTs in vitro18. Despite these observations, whether CENP-E plays a physiological role at the kinetochore-MT interface of congressed chromosomes and the possible underlying molecular mechanisms for these findings remain controversial.
Mechanochemical enzymes like kinesins transport intracellular cargos by walking along MT tracks. The well-studied kinesin-1, a plus-end-directed motor, carries axonal organelles towards MT ends, where the motor dissociates from the MT and unloads its cargo19. However, the chromosomal cargo carried by CENP-E kinesin is unusual; after the kinetochore has encountered the ends of the MT tracks, it does not detach but remains stably associated with the tips of MTs20,21. The nature of the connections that link the kinetochores of congressed chromosomes and MT ends is not well understood, but these links are known to permit exchange of tubulin dimers at the MT ends, implying that each kinetochore can processively track dynamic MT ends20,22. This is a remarkable property because the kinetochore maintains its attachment to the site where tubulin dimers are being added or lost on the MT polymer. Only two proteins that exhibit bi-directional tip-tacking in vitro have thus been identified23–25. Here we examine whether the CENP-E kinesin can facilitate such end-on MT attachments.
To investigate a possible contribution of CENP-E to linking MT plus ends to the kinetochores on congressed chromosomes we used a specific small molecule inhibitor GSK-923295, which locks CENP-E in a “rigor” MT-bound state13. HeLa cells were arrested in metaphase with a proteasome inhibitor, which was then replaced with the CENP-E inhibitor or DMSO as a control (Fig. 1a). In control metaphase cells CENP-E was clearly visible at the kinetochores (Fig. 1b); chromosome alignment was maintained in all but 1 out of 50 cells and anaphase started ~30 min after removing the proteasome inhibitor (Fig.1c, d). Addition of GSK-923295 to metaphase cells caused a marked depletion of the kinetochore-localized CENP-E (Fig. 1e), probably due to the passive transport of the rigor bound motor by poleward MT flux6. Importantly, in ~50% of CENP-E-inhibited cells there was a clear loss of chromosome alignment (3.6 chromosome pairs per cell). One of the sister kinetochores on such pairs appeared to assume MT-lateral binding, and moved toward the pole (Fig. 1e, lower inset). Subsequently, these chromosomes accumulated at the poles and anaphase was delayed (Video S1), the hallmark features of the loss of CENP-E function9,11,17. These results clearly demonstrate that CENP-E contributes not only to the congression of pole-proximal chromosomes5,7, but it also continues to be motor active at the kinetochores of congressed chromosomes.
The above finding prompted us to reconstitute CENP-E interactions with MTs in vitro using purified proteins. To examine transport properties of the previously uncharacterized wild type version of CENP-E, thereafter called full length (FL) CENP-E, we used total internal reflection fluorescence (TIRF) microscopy to visualize a C-terminal fusion of it to GFP (Fig. 2a,b). Many encounters between single FL molecules and coverslip-attached, Taxol-stabilized MTs led to short-lived diffusive motions, suggesting auto-inhibition of soluble FL CENP-E26,27 (Fig. 2c, S1a-d). However, some FL molecules in our preparations lacked such inhibition and moved unidirectionally and processively, similar to a truncated (TR) CENP-E, in which the motor domains were dimerized with a shortened coiled-coil stalk6,28 (Fig. 2a). These proteins were then conjugated by their C-termini to 0.5μm beads, and laser tweezers were used to launch these beads on MT walls (Figs. S1e-g, Video S2). A significantly larger fraction of processive motions was observed with beads coated with FL CENP-E vs. what was seen with the soluble bead-free molecules (Fig. S1h), suggesting that conjugating the tail of FL CENP-E to a bead cargo partially relieves the auto-inhibition, as in kinesin-129. Detailed quantification of the single molecule and bead motility in these assays revealed that the MT wall-dependent transport by FL motor is highly similar to that of TR CENP-E (Table S1)8,28,30.
We then analyzed what happens when CENP-E walks to the ends of MT tracks. Upon reaching the plus tips of stable MTs, both FL and TR CENP-E constructs paused at the tip for 2-4 sec before detaching8, significantly longer than 0.3±0.1sec (Mean±SEM, n=253) for kinesin-1 studied under identical conditions (Fig. S2a and Table S2). This finding may explain an increase in the rate of MT polymerization, which was observed previously with human truncated CENP-E motor in the presence of Taxol31. To examine how CENP-E interacts with the physiologically relevant, dynamic MT ends, we grew non-stabilized polymers from coverslip-attached MT seeds (Fig. 3a). Both FL and TR CENP-E proteins walked fast enough to catch up with the growing MT end. Interestingly, the TR molecules detached from these ends more rapidly than from the tips of Taxol-stabilized MTs (Figs. 3b, S2; Video S3). However, a large fraction of FL CENP-E molecules did not dissociate from the polymerizing ends and continued to move with MT elongation, which was 15 times slower than the motor's walking rate (Figs. 3c,d, S3). This marked slowing is unlikely to result from the different nucleotide composition of the growing tip vs. MT wall because FL CENP-E moved only slightly slower on the GMPCPP vs. GDP-containing lattices (Fig. S3c). A slower motility on GMPCPP-containing segments was also observed for TR CENP-E, yet it failed to track processively with the polymerizing tip (Fig. S2c).
About 70% of FL motors that walked into a disassembling MT tip also did not dissociate but moved backwards in the direction opposite to the motor-dependent motility, while following the rapidly disassembling MT (Fig. 3d; Videos S4, S5). By quantifying GFP intensity of the tip-tracking complexes we found that many contained only 2 GFP molecules and therefore were dimers of CENP-E (Figs. 3e, S3 e-g). The tip-tracking complexes sometimes appeared to diffuse near the tip. This behavior was more evident at polymerizing tips, presumably because polymerization is 10-times slower than depolymerization (Figs. 3d, Video S6). FL CENP-E dimers remained associated with growing and shortening tips for 17.9±1.3 and 11.6±1.4 sec, respectively, resulting in the travel lengths of 0.3±0.1 and 2.1±0.3 μm (Fig. S4a-d; Table S2). Small teams of FL molecules tracked even better, sometimes remaining at the tip for several rounds of MT growth and shortening (Video S7). These brighter complexes tended to slow MT depolymerization (Fig. S3b), but single tip-tracking dimers did not affect significantly either the rates of growth and shortening, or catastrophe frequency (Fig. 3f). Thus, CENP-E has a capacity to track processively with both assembling and disassembling MTs at a single molecule level, a property not shared by previously studied kinesins.
CENP-E has been observed to localize to the plus ends of spindle MTs in human cells, consistent with its tip-tracking in vivo15. Furthermore, prior work implicated CENP-E in assisting motions of isolated mammalian chromosomes at the ends of depolymerizing MTs in vitro18. To test directly whether purified FL CENP-E can couple MT dynamics to cargo motion, we used segmented MTs, an in vitro approach we previously developed to study MT depolymerization-dependent motions32 (Fig. 4a). With laser tweezers, CENP-E-coated beads were brought to the walls of MTs, transiently stabilized by caps made from GMPCPP-containing Rhodamine-labeled tubulin. These beads walked toward the capped plus-ends, mimicking chromosomal transport during congression. When MT depolymerization was triggered by photo-ablating the stabilizing caps, almost all FL CENP-E-coated beads followed the shortening MT ends (Fig. 3b-e), demonstrating the ability of CENP-E to couple beads motions with MT disassembly. Such coupling required the non-motor domains of CENP-E as TR CENP-E-coated beads failed to track (Videos S8, S9).
To test a possible role for CENP-E in linking kinetochores and dynamic MT ends, we designed an experiment to trigger chromosome transport by depolymerizing MTs in vivo33 (Fig. 5a). Monopolar spindles were induced in HeLa cells that expressed the kinetochore marker Mis12-GFP. After chromosomes assumed their positions at the plus tips of astral MTs, the CENP-E motor was inhibited with GSK-923295, after which MT depolymerization was triggered by nocodazole (Fig. 5b). Live imaging of control cells with no CENP-E inhibitor revealed that nocodazole-induced MT shortening produced chromosome motion toward the unseparated poles (Figs. 5c,d). Inhibiting CENP-E, however, greatly reduced the ability of chromosomes to move in conjunction with MT disassembly (Video S10).
To rule out that the CENP-E inhibitor directly affected the MT stability, cells were subjected to the same treatments (as in Fig. 5b), but their spindles were visualized with tubulin-EYFP. MT depolymerization commenced in <1 min in all cells treated with nocodazole, but adding GSK-923295 alone had no effect (Fig. S5). No gross changes in MT asters were seen in cells that were fixed and immunostained 25 min after adding GSK-923295, as judged by spindle tubulin intensity (Fig. S6). The diameters of MT asters were slightly larger in GSK-923295 treated cells, further indicating that this inhibitor did not induce MT disassembly (Fig. S6c). Next, cells were pretreated with GSK-923295 for 5 min to deplete CENP-E from the kinetochores, then additionally treated with nocodazole and cold to promote MT disassembly. In ~60% of these cells some chromosomes were left at the periphery with no attached cold-stable MT fibers (Figs. 5e,f; S7a). This is a significantly larger fraction than when nocodazole was used with no CENP-E inhibitor, consistent with live imaging results. Furthermore, the GSK-923295 pretreated cells had fewer tubulin-containing MT remnants than the cells with CENP-E-depleted kinetochores (Fig. S6a,d). Similar results were also seen when CENP-E was disrupted by RNAi in another human cell line and similar experiments were done without GSK-923295 (Figs. 5g,h; S7b). We conclude that presence of CENP-E motor at the kinetochores promotes their ability to maintain stable connections with disassembling MT ends.
To dissect the molecular mechanisms underlying the processive tip-tracking we tested two specific hypotheses. First, we asked if tip-tracking could result from the increased affinity of CENP-E to MT tips vs. walls. With a fluorescence-based MT-pelleting assay we measured CENP-E binding to GDP- and GMPCPP-containing MTs, mimicking the growing MT tips: the apparent dissociation constants were 41±4 nM and 68±23 nM, respectively (Fig. 6a). Binding affinity to the depolymerizing MT tips, as measured with tubulin spirals that are similar in structure to the curved tubulin protofilaments34, was 133±22 nM. Thus, CENP-E shows no significant preference to the tip-mimicking structures, suggesting that its bi-directional tip-tracking is unlikely to results from different affinities.
Since TR CENP-E was unable to track MT ends processively, we next investigated if the C-terminal tail of CENP-E was involved (Fig. 2a). FL bound stronger to Taxol-stabilized MTs than the TR protein (144±54 nM; Fig. 6a), consistent with the presence of a MT-binding site in its tail26,35. We expressed and purified a C-terminal CENP-E fragment fused with GFP (Tail, Fig. 6b). This fragment lies distally to the kinetochore-targeting sequence of CENP-E26,36, so the tails of kinetochore-bound CENP-E molecules may be positioned to interact directly with kinetochore MTs in vivo. Purified Tail molecules bound and diffused rapidly on MTs but failed to follow the dynamic tips, indicating that tail was not the sole determinant of the bi-directional tip-tracking (Fig. 6c-f). Interestingly, statistical analysis of motions of the Tail at the dynamic MT ends has revealed that only 1 out of 10 encounters between the Tail and dynamic MT end resulted in Tail's detachment (see Materials and Methods), with the majority of Tail molecules bouncing back from the tip and continuing to diffuse along the MT wall, as if “reflected” by the tip (Fig. 6 g,h).
Based on the above results we then hypothesized that even though neither the tail nor the motor domain could track the dynamic MT ends on their own, a combination of their molecular properties produced a collective tip-tracking behavior. We tested this hypothesis in a mechanistic and quantitative way with a mathematical model based on the detailed kinetic schemes (Figs. 7a,b). The model describes molecular interactions between the MT wall, tip, and the motor and tail domains connected together via a worm-like tether (see Supplementary Note for detail description of model assumptions and Table S3 for parameter values). This quantitative model recapitulated the major results of our in vitro studies with TR and FL CENP-E, including rapid dissociation of the TR motor from dynamic MT ends and the ability of FL CENP-E to tip-track processively (Figs. 7c-f). It also provided a physical mechanism for the cooperative action of motor and tail domains by demonstrating that FL CENP-E can track bi-directionally by repeating the cycles of plus end-directed walking and the tail-mediated diffusion of the MT wall-tethered motor (Videos S11, S12). The tail-MT wall association, although transient, keeps the tail-tethered motor heads in the vicinity of MT. We estimate that tethered motor heads re-associate with the wall within a millisecond, so the tail serves as a safety leash for the motor heads, which dissociate rapidly and repeatedly from the tip. Remarkably, this behavior in silico does not rely on difference(s) between assembling and disassembling ends, explaining how CENP-E tracks with both types of ends.
The proposed “tethered motor” mechanism of tip-tracking is strongly supported by our experimental observations of diffusive motions of the tip-tracking FL molecules, as evidenced by the ragged appearance of kymographs (Fig. 8 a-c). Several examples of visible excursions at both elongating and shortening MT ends are shown in Fig. S3d, and the smaller diffusive motions at the tips were common (e.g. Video S6). We challenged our model by testing experimentally its two key predictions. First, we examined tip-tracking in the presence of the non-hydrolysable ATP analog, AMPPNP, which induces strong MT-binding. Consistent with the model's prediction, the inhibited FL CENP-E failed to follow the disassembling MT tips (Fig. 8d), demonstrating that the motor's walking is required for processive tip-tracking. Second, we recapitulated the wild-type FL CENP-E motions by artificially joining the non-tracking TR CENP-E and Tail proteins. Qdots coated with the mixture of these proteins exhibited robust walking and also moved processively with both assembling and disassembling MT ends (Fig. 8e). Thus, tail and motor domains cooperate together to provide processive tip association.
Here we describe the results of the integrated study of motility of CENP-E kinesin and its physiological implications using in vivo, in vitro and in silico approaches. Experiments in two human cell lines show that kinetochore-MT attachments in disassembling monopolar spindles are greatly destabilized when CENP-E function is perturbed with either a specific inhibitor or RNAi depletion. Furthermore, we establish a continuing in vivo requirement for CENP-E motor activity at the kinetochores of congressed and bi-oriented chromosomes. Together with our discovery of CENP-E's ability to associate with the ends of dynamic MTs in vitro, these findings suggest a previously unrecognized function for this essential kinesin in accurate chromosome segregation. We propose that in addition to its established role in transporting polar chromosomes, CENP-E also facilitates the association between kinetochore and dynamic MT ends. In vitro, CENP-E kinesin can convert from a lateral transporter into a MT tip-tracker, so a similar activity may contribute to the formation of end-on MT attachments for the chromosomes that congress via a kinetochore-fiber-independent pathway7. Although a significant fraction of CENP-E protein leaves kinetochores after congression, CENP-E is clearly present at the kinetochores of bi-oriented chromosomes14,15, and our results in vivo establish the physiological role of this CENP-E population in maintaining end-on MT attachments. These findings are supported by previous studies in different cells systems including Drosophila37, mouse11 and human cells16, and they also provide a molecular mechanism to explain a reduced number of kinetochore MTs on congressed chromosomes in CENP-E depleted cells11,16.
Since we have shown that a single dimer of CENP-E can maintain long-lasting association with a dynamic MT end in vitro without significantly affecting MT dynamics, we also propose that a major component of the mechanism underlying CENP-E-mediated stabilization of kinetochore-MT attachments is a direct one, provided by its ability to serve as a “mobile” molecular bond between the kinetochore and dynamic kinetochore-MT tip. Future work, however, is required to examine whether the kinetochore-bound CENP-E can also affect dynamics of kinetochore MTs, since multiple CENP-E molecules may act in concert at kinetochores. Furthermore, in the context of live cells the tip-tracking by CENP-E may promote kinetochore-MT attachment not only directly but also in conjunction with additional activities from other associated proteins, such as CLASP1/238,39 or PP1 phosphatase8.
Our work also provides a detailed molecular mechanism to explain how CENP-E can track MT tips processively and bi-directionally. The autonomous tip-tracking that we have uncovered for CENP-E is distinct from that of proteins like EB1. Despite being widely referred to as a plus-tip-tracking protein, the individual molecules of EB1 do not associate continuously with growing MTs and tip-tracking is not processive40. Only two proteins, XMAP21523 and Dam1 oligomers25,41,42, neither with motor activity, have previously been described to track dynamic MT tips processively and bi-directionally at a single molecule level. Members of the kinesin-8 family, the plus-end-directed Kif18A and Kip3, can associate processively with the growing MT tip in vitro, assisted by their C-terminal tails43–46. These motors, however, fail to track disassembling MTs. The molecular mechanism of their unidirectional plus-end-tracking is not known, so it may include the affinity-based interactions specifically at the assembling tips. Alternatively, kinesin-8 may tip-track with the growing MTs by the mechanism we propose here for CENP-E. Our computational model provides insight into protein's ability to tip-track uni- vs. bi-directionally by emphasizing a complex interrelationship between the rates of protein diffusion and MT dynamics47. The reported diffusion of the Kif18A tail is 100-times slower than that of the CENP-E tail43,45. In silico, motors with both tails can travel with the elongating MT end quite well because both tails diffuse rapidly relative to the slow rate of MT assembly (Fig. 8f). However, the slowly diffusing tail is predicted to dissociate sooner from the shortening MT ends because MT disassembly is rapid enough to catch up repeatedly with the molecule that diffuses slowly “in front” of the wave of tubulin depolymerization. Each of these encounters may cause a stochastic loss of the terminal tubulin dimer together with the bound tail molecule, so the slower diffusing Kif18A tail is not a good tether for the shortening MT end. It remains to be seen if the “tethered motor” mechanism that we propose is employed by other MT-dependent motors, or this specialization is unique to kinetochore-localized kinesin-7.
We thank members of Grishchuk and Ataullakhanov labs for stimulating discussions and technical assistance, A. Korbalev for help with the model videos, M. Porter for a kind gift of axonemes, M. Ostap, M. Lampson and J.R. McIntosh for critical reading of the manuscript, E. Ballister and M. Lampson for assistance with live cell imaging. We thank University of California, San Diego, Neuroscience Microscopy Shared Facility (P30 NS047101), and J.R. McIntosh for help with the initial phase of this work (GM 033787). This work has been supported by grants from the NIH to E.L.G. (R01-GM098389) and to D.W.C. (R01-GM29513), and grants to F.I.A from Russian Fund for Basic Research (12-04-00111-a) and Presidium of Russian Academy of Sciences (Mechanisms of the Molecular Systems Integration and Molecular and Cell Biology programs). B.V. has been supported by a postdoctoral fellowship from the Human Frontiers Science Program. E.L.G. is a Kimmel Scholar; her work is supported in part by the Pennsylvania Muscle Institute. D.W.C. receives salary support from the Ludwig Institute for Cancer Research.
Author contributions: N.G. performed in vitro experiments and mathematical modeling; B.V. and A.K. performed in vivo experiments; B.V. and Y.K. purified proteins; N.G., B.V., F.I.A., D.W.C. and E.L.G. designed research, analyzed data and wrote the paper.
Competing financial interests: The authors declare no competing financial interests.