RESUMEN
The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of kinetochore-microtubule attachments that come under tension due to opposing pulling forces exerted on sister kinetochores by dynamic microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to kinetochore-microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 kinetochore complex that strengthens its attachment to microtubule tips. Strikingly, Stu2 can either stabilize or destabilize kinetochore attachments, depending on the level of kinetochore tension and whether the microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of microtubule dynamics. Altogether, our results demonstrate how a kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation.
Asunto(s)
Segregación Cromosómica , Cinetocoros/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Fenómenos Biomecánicos , Humanos , Proteínas Nucleares/fisiología , Unión ProteicaRESUMEN
Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
Asunto(s)
Proteínas Cromosómicas no Histona , Nucleosomas , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Centrómero/genética , Centrómero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.
Asunto(s)
Cinetocoros , Proteínas de Saccharomyces cerevisiae , Humanos , Cinetocoros/metabolismo , Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microtúbulos/metabolismo , Segregación Cromosómica/genética , Ploidias , AneuploidiaRESUMEN
Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical, and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide the combination of plasticity and strength that allows kinetochores to maintain load-bearing tip attachments during both microtubule assembly and disassembly.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologíaRESUMEN
Dynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle in close proximity to the bud neck, such that, at anaphase onset, the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements within the mother cell, thus ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Proteínas de Saccharomyces cerevisiae , Dineínas/genética , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.
Asunto(s)
Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Humanos , Cinetocoros/química , Microtúbulos/metabolismo , Modelos Biológicos , Fosforilación , Unión Proteica , Saccharomycetales/metabolismoRESUMEN
Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation.
Asunto(s)
Aurora Quinasas/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Aurora Quinasas/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by â¼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Oxígeno/metabolismo , Carbocianinas/metabolismo , Línea Celular , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Fluorescencia , Colorantes Fluorescentes/metabolismo , Glucosa Oxidasa/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Cetoácidos/metabolismo , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , NAD/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fotoblanqueo , Receptor PAR-2/metabolismoRESUMEN
Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore-microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular , Segregación Cromosómica , Proteínas del Citoesqueleto , Humanos , Cinetocoros/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/genética , Proteínas Nucleares/genética , Unión ProteicaRESUMEN
Metaphase chromosome positioning depends on Kif18A, a kinesin-8 that accumulates at and suppresses the dynamics of K-MT plus ends. By engineering Kif18A mutants that suppress MT dynamics but fail to concentrate at K-MT plus ends, we identify a mechanism that allows Kif18A to accumulate at K-MT plus ends to a level required to suppress chromosome movements. Enrichment of Kif18A at K-MT plus ends depends on its C-terminal tail domain, while the ability of Kif18A to suppress MT growth is conferred by the N-terminal motor domain. The Kif18A tail contains a second MT-binding domain that diffuses along the MT lattice, suggesting that it tethers the motor to the MT track. Consistently, the tail enhances Kif18A processivity and is crucial for it to accumulate at K-MT plus ends. The heightened processivity of Kif18A, conferred by its tail domain, thus promotes concentration of Kif18A at K-MT plus ends, where it suppresses their dynamics to control chromosome movements.
Asunto(s)
Cinesinas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Posicionamiento de Cromosoma , Células HeLa , Humanos , Cinesinas/genéticaRESUMEN
Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore-microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Complejos Multiproteicos/genética , Mutación , Estructura Terciaria de ProteínaRESUMEN
Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this angler's trick, operating at kinetochores, ensures accuracy during mitosis: the mitotic spindle attaches randomly to chromosomes and then correctly bioriented attachments are stabilized due to the tension exerted on them by opposing microtubules. Incorrect attachments, which lack tension, are unstable and release quickly, allowing another chance for biorientation. Stabilization of molecular interactions by tension also occurs in other physiological contexts, such as cell adhesion, motility, hemostasis, and tissue morphogenesis. Here, we review models for the stabilization of kinetochore attachments with an eye toward emerging models for other force-activated systems. Although attention in the mitosis field has focused mainly on one kinase-based mechanism, multiple mechanisms may act together to stabilize properly bioriented kinetochores and some principles governing other tension-sensitive systems may also apply to kinetochores.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis , Animales , Aurora Quinasas/metabolismo , Fenómenos Biomecánicos , Humanos , Modelos BiológicosRESUMEN
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
Asunto(s)
Cromosomas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fenómenos Biomecánicos , Cinética , Imitación Molecular , Complejos Multiproteicos/metabolismo , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.
Asunto(s)
Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aurora Quinasa B , Aurora Quinasas , Proteínas del Citoesqueleto , Humanos , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mutación/genética , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , Cinetocoros/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Fenómenos Biomecánicos , Proteínas de Ciclo Celular/genética , Microesferas , Proteínas Asociadas a Microtúbulos/genética , Pinzas Ópticas , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
RESUMEN
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
RESUMEN
Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
RESUMEN
During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.