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Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics., During cell division, it is currently unclear how kinetochores transit from lateral microtubule attachment to durable association to dynamic microtubule plus ends. Here, using in vitro reconstitution and computer modeling, the authors provide biophysical mechanism for microtubule end-conversion driven by two kinetochore components, CENP-E and Ndc80 complex

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are ill-understood. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

A mathematical model is used to analyze the impact of the indiscriminate kinetochore microtubule turnover and the back-to-back kinetochore geometry on chromosome biorientation during mitosis. The authors show that mammalian kinetochore operates in a near-optimal regime, whereby these two features provide a significant error-correction activity., Accuracy of chromosome segregation relies on the ill-understood ability of mitotic kinetochores to biorient, whereupon each sister kinetochore forms microtubule (MT) attachments to only one spindle pole. Because initial MT attachments result from chance encounters with the kinetochores, biorientation must rely on specific mechanisms to avoid and resolve improper attachments. Here we use mathematical modeling to critically analyze the error-correction potential of a simplified biorientation mechanism, which involves the back-to-back arrangement of sister kinetochores and the marked instability of kinetochore–MT attachments. We show that a typical mammalian kinetochore operates in a near-optimal regime, in which the back-to-back kinetochore geometry and the indiscriminate kinetochore–MT turnover provide strong error-correction activity. In human cells, this mechanism alone can potentially enable normal segregation of 45 out of 46 chromosomes during one mitotic division, corresponding to a mis-segregation rate in the range of 10−1–10−2 per chromosome. This theoretical upper limit for chromosome segregation accuracy predicted with the basic mechanism is close to the mis-segregation rate in some cancer cells; however, it cannot explain the relatively low chromosome loss in diploid human cells, consistent with their reliance on additional mechanisms.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. We thank F. I. Ataullakhanov for help with the laser trap and data analysis; A. Kiyatkin, V. Mustyatsa, M. Molodtsov, A. Gautreau, G. Lakisic, and M. Barisic for technical assistance; and members of our laboratories for stimulating discussions. This work was supported by National Institutes of Health grant R01-GM098389 and RSG-14-018-01-CCG from the American Cancer Society to E.L.G.; by the Institut Curie, the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the L'Agence Nationale de la Recherche (ANR) award ANR-12-BSV2-0007, INCA_6517, ANR-10-LBX-0038, part of the IDEX Idex PSL, ANR-10-IDEX-0001-02 PSL to C.J.; and Fundacao Luso-Americana para o Desenvolvimento (FLAD) Life Science 2020 and PRECISE grant from the European Research Council to H.M. A.V.Z. is supported by the RAS Presidium Grants "Mechanisms of the Molecular Systems Integration," " Molecular and Cell Biology programs," and Russian Fund for Basic Research Grant 12-04-00111-a and 13-00-40188. R.S.S. is supported by a fellowship from the Programa Graduado em Areas da Biologia Basica e Aplicada (GABBA) PhD program from the University of Porto. A.L.P. is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a Ciencia e a Tecnologia of Portugal. M.B., R.S.S., S.K.T., M.M.M., C.J., E.L.G., and H.M. designed the experiments; M.B. performed all experiments in cells; M. M. M. established and performed the tubulin purification protocol from HeLa cells; R.S.S. performed single-molecule experiments; S.K.T. performed force measurements; A.L.P. provided reagents; all authors analyzed data; H.M., E.L.G., and M.B. wrote the paper, with contributions from all authors; H.M. conceived and coordinated the project. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests.

Both N- and C-terminal microtubule (MT)-binding domains of CENP-F can follow depolymerizing MT ends while bearing a significant load, and the N-terminal domain prefers binding to curled oligomers of tubulin relative to MT walls by approximately fivefold, suggesting that CENP-F may play a role in the firm bonds that form between kinetochores and the flared plus ends of dynamic MTs., Firm attachments between kinetochores and dynamic spindle microtubules (MTs) are important for accurate chromosome segregation. Centromere protein F (CENP-F) has been shown to include two MT-binding domains, so it may participate in this key mitotic process. Here, we show that the N-terminal MT-binding domain of CENP-F prefers curled oligomers of tubulin relative to MT walls by approximately fivefold, suggesting that it may contribute to the firm bonds between kinetochores and the flared plus ends of dynamic MTs. A polypeptide from CENP-F’s C terminus also bound MTs, and either protein fragment diffused on a stable MT wall. They also followed the ends of dynamic MTs as they shortened. When either fragment was coupled to a microbead, the force it could transduce from a shortening MT averaged 3–5 pN but could exceed 10 pN, identifying CENP-F as a highly effective coupler to shortening MTs.

Accurate regulation of kinetochore–microtubule affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which NDC80–microtubule bonds reorganize dynamically in response to the number and stability of microtubule attachments., Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore–MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore–MT affinity. In contrast, modeling the kinetochore interface with repetitive MT binding sites predicts a switchlike response. To reconcile these findings, we hypothesize that interactions between NDC80 complexes and MTs are not constrained, i.e., the NDC80 complexes can alternate their binding between adjacent kinetochore MTs. Experiments using cells with phosphomimetic Hec1 mutants corroborate predictions of such a model but not of the repetitive sites model. We propose that accurate regulation of kinetochore–MT affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which the NDC80–MT bonds reorganize dynamically in response to the number and stability of MT attachments.

Chromosome segregation during mitosis is mediated by spindle microtubules that attach to chromosomal kinetochores with strong yet labile links. The exact molecular composition of the kinetochore–microtubule interface is not known but microtubules are thought to bind to kinetochores via the specialized microtubule-binding sites, which contain multiple microtubule-binding proteins. During prometaphase the lifetime of microtubule attachments is short but in metaphase it increases 3-fold, presumably owing to dephosphorylation of the microtubule-binding proteins that increases their affinity. Here, we use mathematical modeling to examine in quantitative and systematic manner the general relationships between the molecular properties of microtubule-binding proteins and the resulting stability of microtubule attachment to the protein-containing kinetochore site. We show that when the protein connections are stochastic, the physiological rate of microtubule turnover is achieved only if these molecular interactions are very transient, each lasting fraction of a second. This “microscopic” time is almost four orders of magnitude shorter than the characteristic time of kinetochore–microtubule attachment. Cooperativity of the microtubule-binding events further increases the disparity of these time scales. Furthermore, for all values of kinetic parameters the microtubule stability is very sensitive to the minor changes in the molecular constants. Such sensitivity of the lifetime of microtubule attachment to the kinetics and cooperativity of molecular interactions at the microtubule-binding site may hinder the accurate regulation of kinetochore–microtubule stability during mitotic progression, and it necessitates detailed experimental examination of the microtubule-binding properties of kinetochore-localized proteins.

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule–ring–bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.

Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division. DOI: http://dx.doi.org/10.7554/eLife.10644.001, eLife digest Cell division is a highly organized process that involves a series of major changes. First, the cell’s chromosomes are copied and arranged at the middle of the cell. Then, the pairs of copied chromosomes are separated and pulled towards opposite ends of the cell and, finally, the cell splits in two. These steps are mainly regulated by modifications to proteins, and enzymes called protein kinases play an important role because they add phosphate groups to, or phosphorylate, so-called 'substrate' proteins to change their activities. Other enzymes called phosphatases are also important because they remove the phosphate groups from the substrates to reverse the effects. The kinase Aurora B is required for several steps during cell division and has been widely studied. This kinase is enriched in specific locations within the cell, for example at the centromere regions of the chromosomes as they line up at the cell’s center. However, Aurora B phosphorylates substrates located at distant sites on the chromosome, with less phosphorylation at sites farther from the centromere. The level of phosphorylation also changes as chromosomes become aligned. Aurora B can activate itself and this ability was suspected to help this spatiotemporal regulation. However, it was not clear how the observed gradients of kinase activity might form. Zaytsev, Segura-Peña et al. set out to answer this question by first mixing in a test tube purified Aurora B and an inhibitory phosphatase. This revealed that this kinase-phosphatase system is 'bistable', meaning that it has two stable states, low or high kinase activity, and that these states could switch in response to small changes in enzyme concentrations. Further experiments showed that this system has a kind of memory such that the level of activity (low or high) persists for a range of concentrations and depends on the system’s prior history. Zaytsev, Segura-Peña et al. then showed that both of these properties, the two stable states and the memory, exist in dividing human cells, and then went on to develop a mathematical model of how such bistability could set up gradients of Aurora B kinase activity. At the sites of highest concentration at the centromere, Aurora B can overcome inhibition by phosphatase and activates itself, as in the test tube. This activity spreads to more distant locations as active kinase molecules activate neighboring kinase molecules, establishing the areas with a high state of activity. As the local Aurora B concentration decreases further from the centromere, the phosphatase switches Aurora B into the low activity state, establishing a steep gradient of kinase activity in a region where its substrates that are important for chromosome segregation are located. Importantly, the shape and location of this gradient are predicted to depend on forces that stretch the lined chromosomes apart, offering a plausible mechanism to explain phosphorylation changes in response to tension. These theoretical insights and experimental approaches could be used to study other coupled kinase-phosphatase systems. DOI: http://dx.doi.org/10.7554/eLife.10644.002

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

This work defines the biophysical mechanism of phosphoregulation of microtubule binding by the kinetochore NDC80 complex. Conformational plasticity of the disordered tail of its Hec1 subunit integrates outputs from multiple phosphorylations to serve as a rheostat, providing a new paradigm for accurate regulation of microtubule-binding affinity., Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Proper regulation of dynamic interactions between microtubules and chromosomal kinetochores is vitally important for accurate cell division, but little is known about how such regulation is achieved. Here, we used single molecule and quantitative fluorescence microscopy approaches to dissect microtubule binding of the multisubunit NDC80 complex, a core kinetochore microtubule-binding component. Human GFP-labeled NDC80 proteins were designed with up to nine phospho-mimetic aspartates, all located in the N-terminal tail of Hec1 subunit in a different combinations. Previous studies have suggested that one set of these phospho-residues controls primarily the affinity of NDC80 binding to microtubule, while the other set regulates binding cooperativity. We provide evidence, however, that phosphorylation of Hec1 tail has little impact on the cooperativity of NDC80 binding. In contrast, the microtubule binding affinity, as estimated from the residency time and diffusion coefficient of single NDC80 complexes, is highly sensitive to Hec1 tail phosphorylation. The binding energy decreases linearly at about 0.3 kBT per added phosphoresidue regardless of its specific position in Hec1 tail. To gain insight into such unusual regulatory behavior we have carried out molecular dynamics simulations of the disordered Hec1 tail in the context of microtubule-bound NDC80 complexes. These theoretical simulations revealed multiple conformations of the Hec1 tail bound to microtubule. Interestingly, the contact area between Hec1 tail and microtubule decreases proportionally to the number but not the locations of phospho-mimetic mutations in Hec1 tail. We propose that the disordered nature of NDC80 tail renders its microtubule binding insensitive to the exact location of phosphorylated residue, thereby enabling the gradual tuning of kinetochore-microtubule binding affinity during mitosis.

The stability of kinetochore-microtubule (KMT) attachments is finely tuned to drive different mitotic processes. This regulation involves phosphorylation of NDC80 complex, a major component of the MT-binding interface at kinetochores. The fundamental question of how the number and stability of KMT attachments is regulated by NDC80 phosphorylation remains unanswered. The Hec1 subunit of the NDC80 complex has an unstructured “tail,” which is required for KMT attachment in vivo and contributes to the NDC80 complex-MT binding in vitro. This tail is an established target for Aurora kinases and has 9 mapped phosphorylation sites. To understand how phosphorylation of the Hec1 tail affects MT-binding characteristics of single NDC80 complexes we expressed and purified NDC80Bonsai complexes with different number of phospho-mimetic mutations in Hec1 tail. With TIRF microscopy we found that Hec1 tail phosphorylation leads to a graded increase in NDC80 diffusion and the shortening of its MT residency time, but cooperativity of NDC80-MT binding is only weakly affected by Hec1 tail phosphorylation. To understand physiological relevance of the phosphoregulation of NDC80 complexes we used computational approaches to model kinetochore-MT interface containing multiple NDC80 molecules. We show that the behavior of such interface strongly depends on the spatial organization of the NDC80 complexes. KMT interface that contained “repetitive sites” of NDC80 complexes greatly amplified the relatively small, phosphorylation-induced changes in the residency time of single NDC80. However, the KMT interface that contained a dynamic “lawn” of un-clustered and uncoordinated NDC80 complexes exhibited a graded response to phosphorylation and produced an excellent fit to our data with cells in prometaphase and metaphase. We conclude that incremental phosphorylation of NDC80 complexes drives the graded regulation of kinetochore-microtubule affinity during mitotic progression.

The accuracy of chromosome segregation relies on the remarkable ability of mitotic kinetochores to bi-orient, whereby sister kinetochores form microtubule attachments to opposing spindle poles. Since the probability of forming erroneous attachments vastly exceeds the chance of attaching correctly, several mechanisms have been proposed to explain how kinetochores avoid and resolve these errors. Here, we use quantitative molecular-mechanical modeling of the kinetochore-microtubule interface to evaluate these factors and determine their respective roles. Our analysis defines several key features that ensure expedient error correction. First, geometric constraints, wich bias orientation of paired sister kinetochores such that they preferentially face opposite poles, contribute to proper attachments, but they are not sufficient to provide error-free segregation. Second, two aspects of Aurora B kinase-dependent regulation play significant and distinct roles in establishing and maintaining correct microtubule-attachments: 1) Its ability to promote the rapid turn-over of all kinetochore-microtubule attachments, not just those that are inappropriately attached, and 2) phosphor-regulation of microtubule affinity of spatially-distributed factors in a manner that depends on inter-kinetochore tension. However, a combination of geometric constraints and the error-resolving activities of Aurora B are not sufficient for a fully robust error correction. To solve this problem we hypothesize that the individual microtubule binding sites behave semi-autonomously such that intra-kinetochore tension arises locally in a flexible kinetochore meshwork. Indeed, when such a meshwork is added to the model, kinetochore bi-orientation and expedient error correction occur in a highly reproducible, deterministic way over a significant range of system parameters and even when cells are challenged by error-inducing treatments. Our work has generated the first comprehensive quantitative model to explain spatiotemporal self-organization during chromosome segregation and has provided a solid molecular-mechanical basis for the error correction mechanism.

Targeted transport by intracellular motors can be regulated by posttranslational modifications of polymerized tubulins in the microtubule tracks, but little is known about such effects for motors that drive chromosome motions during mitosis. Microtubules that form mitotic spindle are differentially modified at the C-terminal residue of α-tubulin: polymers that point to the spindle equator, but not the astral microtubules, are preferentially detyrosinated. Here we examine the influence of tubulin detyrosination on CENP-E, the kinetochore-localized kinesin-7 that transports pole-proximal chromosomes to the spindle equator. We polymerized purified human tubulins that were fully tyrosinated or detyrosinated, and examined the suitability of these tracks for motility of recombinant GFP-tagged CENP-E motor. Using fluorescence microscopy we show that single molecules of CENP-E walk faster and more processively on detyrosinated microtubules. Moreover, on these tracks the CENP-E motor can generate larger force than on the tyrosinated microtubules, as determined using stationary optical trap. On both types of microtubules CENP-E took 8-nm steps, exhibited similar dwell times and frequencies of backward stepping. However, motor's detachment increased with resisting force faster when CENP-E was walking on tyrosinated microtubules, leading to the detachment from these polymers at on average smaller load, 4.5 pN vs. 6.4 pN for detyrosinated microtubules. The enhanced motility of CENP-E motor on detyrosinated microtubules, most notably its ability to carry a larger load, could potentially explain the targeted transport of mitotic chromosomes toward the spindle equator.