The outer plate in vertebrate kinetochores is a flexible network with multiple microtubule interactions

Intricate interactions between kinetochores and microtubules are essential for the proper distribution of chromosomes during mitosis. A crucial long-standing question is how vertebrate kinetochores generate chromosome motion while maintaining attachments to the dynamic plus ends of the multiple kinetochore MTs (kMTs) in a kinetochore fibre. Here, we demonstrate that individual kMTs in PtK(1) cells are attached to the kinetochore outer plate by several fibres that either embed the microtubule plus-end tips in a radial mesh, or extend out from the outer plate to bind microtubule walls. The extended fibres also interact with the walls of nearby microtubules that are not part of the kinetochore fibre. These structural data, in combination with other recent reports, support a network model of kMT attachment wherein the fibrous network in the unbound outer plate, including the Hec1-Ndc80 complex, dissociates and rearranges to form kMT attachments.     

Comparative autoregressive moving average analysis of kinetochore microtubule dynamics in yeast

To elucidate the regulation of kinetochore microtubules (kMTs) by kinetochore proteins in Saccharomyces cerevisiae, we need tools to characterize and compare stochastic kMT dynamics. Here we show that autoregressive moving average (ARMA) models, combined with a statistical framework for testing the significance of differences between ARMA model parameters, provide a sensitive method for identifying the subtle changes in kMT dynamics associated with kinetochore protein mutations. Applying ARMA analysis to G1 kMT dynamics, we found that 1), kMT dynamics in the kinetochore protein mutants okp1-5 and kip3Delta are different from those in wild-type, demonstrating the regulation of kMTs by kinetochore proteins; 2), the kinase Ipl1p regulates kMT dynamics also in G1; and 3), the mutant dam1-1 exhibits three different phenotypes, indicating the central role of Dam1p in maintaining the attachment of kMTs and regulating their dynamics. We also confirmed that kMT dynamics vary with temperature, and are most likely differentially regulated at 37 degrees C. Therefore, when elucidating the role of a protein in kMT regulation using a temperature-sensitive mutant, dynamics in the mutant at its nonpermissive temperature must be compared to those in wild-type at the same temperature, not to those in the mutant at its permissive temperature.     

The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.     

Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.

During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.     

Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.     

Budding Yeast Kinetochore Proteins,Chl4 and Ctf19, Are Required to Maintain SPB-Centromere Proximity during G1 and Late Anaphase

In the budding yeast, centromeres stay clustered near the spindle pole bodies (SPBs) through most of the cell cycle. This SPB-centromere proximity requires microtubules and functional kinetochores, which are protein complexes formed on the centromeres and capable of binding microtubules. The clustering is suggested by earlier studies to depend also on protein-protein interactions between SPB and kinetochore components. Previously it has been shown that the absence of non-essential kinetochore proteins of the Ctf19 complex weakens kinetochore-microtubule interaction, but whether this compromised interaction affects centromere/kinetochore positioning inside the nucleus is unknown. We found that in G1 and in late anaphase, SPB-centromere proximity was disturbed in mutant cells lacking Ctf19 complex members,Chl4p and/or Ctf19p, whose centromeres lay further away from their SPBs than those of the wild-type cells. We unequivocally show that the SPB-centromere proximity and distances are not dependent on physical interactions between SPB and kinetochore components, but involve microtubule-dependent forces only. Further insight on the positional difference between wild-type and mutant kinetochores was gained by generating computational models governed by (1) independently regulated, but constant kinetochore microtubule (kMT) dynamics, (2) poleward tension on kinetochore and the antagonistic polar ejection force and (3) length and force dependent kMT dynamics. Numerical data obtained from the third model concurs with experimental results and suggests that the absence of Chl4p and/or Ctf19p increases the penetration depth of a growing kMT inside the kinetochore and increases the rescue frequency of a depolymerizing kMT. Both the processes result in increased distance between SPB and centromere.     

Anaphase B precedes anaphase A in the mouse egg

Segregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B). The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.     

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.     

Kinetochore microtubules in crane-fly spermatocytes: untreated, 2 degrees C-treated, and 6 degrees C-grown spindles

We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome-to-pole connections in crane-fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short-term (10-15 min) exposure of spermatocytes to 2 degrees C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2 degrees C followed by recovery at 6 degrees C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2 degrees C-treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6 degrees C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6 degrees C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (greater than 3 microns), and many extended to the polar regions of the spindle. Thus, the crane-fly spindle appears not to be as atypical as it was previously suggested to be.     

Morphological aspects of chromosome spindle fibres inMesostoma: “microtubular fir-tree” structures and microtubule association with kinetochores and chromatin

Summary Bipolarly oriented bivalents in spermatocytes of the turbellarianMesostoma ehrenbergii, displaying fast oscillatory movements in metaphase, were studied with the electron microscope. Kinetochores and chromosome fibres were reconstructed using serial sections cut perpendicular to the spindle axis. Only a small proportion of kinetochore microtubules (kMT) is continuous between the kinetochore and the centrosome. kMTs intermingle with non-kinetochore microtubules (non-kMTs), partly inclined with regard to the kMTs, thus forming a chromosome fibre MT lattice. This resembles the microtubular fir-tree structures (MTFT) described by Bajer and Molè-Bajer inHaemanthus endosperm mitosis. A minimal function of the MTFT may be the anchorage of kMTs in the polar region. Regarding the association of MTs with the chromosome, three types of attachment can be discriminated: (1) normal insertion of kMT plus ends in the kinetochore, (2) penetration of kinetochores and deep insertion in the chromatin, and (3) lateral attachment with kinetochore and chromatin. Lateral association of MTs seems to be mediated by filamentous crossbridges. The observations are discussed in connection with possible behaviour of kMTs during kinetochore movement.Abbreviations kMT kinetochore microtubules - MAP microtubule-associated protein - non-kMT non-kinetochore microtubules - MTFT microtubular fir-tree - PCM pericentriolar material     

Kinetochore microtubule numbers of different sized chromosomes

For three species of grasshoppers the volumes of the largest and the smallest metaphase chromosome differ by a factor of 10, but the microtubules (MTs) attached to the individual kinetochores show no corresponding range in numbers. Locusta mitotic metaphase chromosomes range from 2 to 21 μm, and the average number of MTs per kinetochore is 21 with an SD of 4.6. Locusta meiotic bivalents at late metaphase I range from 4 to 40 μm(3), and the kinetochore regions (= two sister kinetochores facing the same spindle pole) have an average of 25 kinetochore microtubules (kMTs) with an SD of 4.9. Anaphase velocities are the same at mitosis and meiosis I. The smaller mitotic metaphase chromosomes of neopodismopsis are similar in size, 6 to 45 μm(3), to Locusta, but they have an average more kMTs, 33, SD = 9.2. The four large Robertsonian fusion chromosomes of neopodismopsis have an average of 67 MTs per kinetochore, the large number possibly the result of a permanent dicentric condition. Chloealtis has three pairs of Robertsonian fusion chromosomes which, at late meiotic metaphase I, form bivalents of 116, 134, and 152 μm (3) with an average of 67 MTs per kinetochore similar to Locusta bivalents, but with a much higher average of 42 MTs per kinetochore region. It is speculated that, in addition to mechanical demands of force, load, and viscosity, the kMT numbers are governed by cell type and evolutionary history of the karyotype in these grasshoppers.     

Microtubule disorientation in anaphase half-spindles during autosome segregation in crane fly spermatocytes

The region between the kinetochores of syntelically oriented autosomes and the pole in meta- and anaphase of Pales ferruginea spermatocytes was studied by means of serial sections. Microtubule (MT) were counted and measured, and the spindle region was reconstructed by superimposition of successive micrographs. Kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs) interdigitate with one another forming a bundle which is often arrow-shaped due to an inclination of nkMTs (skew nkMTs) with respect to the kinetochore-pole axis. The average length of MT in the bundle decreases towards anaphase while the average number increases. The extent of MT disorder in anaphase half-spindles is higher than in metaphase. The number of kMTs inserted in the kinetochore was found to remain unchanged from meta- to early anaphase. Some of the kMTs become divergent in anaphase. The relative proportion of skew nkMTs within the kMT/nkMT bundle is higher in anaphase. It is proposed that the morphological changes observed to occur from meta- to anaphase are due to fragmentation of kMTs followed by disorientation of the MTs pieces. Some aspects of the physical properties of the half-spindles are discussed.     

Mechanisms of microtubule-based kinetochore positioning in the yeast metaphase spindle

It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells.     

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.     

Molecular architecture of vertebrate kinetochores

Kinetochores form a dynamic interface with the microtubules from the mitotic spindle to achieve accurate chromosome segregation. Multiple proteins are assembled on centromeric DNA to form the kinetochore structure. Recent insights regarding the mechanism of kinetochore formation in vertebrate cells have come from the identification and characterization of kinetochore proteins using a variety of approaches. Constitutive centromere associated network (CCAN) proteins create a platform for kinetochore formation. Subsequently, CCAN proteins recruit outer kinetochore components such as KNL1, the Mis12 complex and the Ndc80 complex (KMN network) that attach to the spindle microtubules, together comprising the functional kinetochore. In this review, we introduce and discuss putative roles of CCAN and KMN proteins during the process of kinetochore formation.     

Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore

Vertebrate kinetochores contain over 50 different proteins organized into three distinct regions: the inner plate, outer plate, and fibrous corona. The present study characterizes numerous precursors of kinetochore assembly in a system free of centromeric chromatin, Xenopus extracts. Hydrodynamic analysis suggests there are a minimum of two monomeric proteins and six pre-assembled complexes that accumulate on centromeres to form the kinetochore. The inner and outer kinetochore assemble from at least two distinct kinetochore complexes containing the proteins Mis12, Zwint, and Ndc80, all of which interact by immunoprecipitation. There is also a network of interactions between the fibrous corona proteins that is dissociated by microtubules. We quantify the number of molecules of specific proteins assembled into a single kinetochore. There are between 800 and 1200 molecules of the measured inner and outer kinetochore proteins, demonstrating that the components in these regions are in similar stoichiometry. In contrast, the measured fibrous corona proteins are present at 250-300 molecules per kinetochore. Zwint, but not Mis12, requires the Ndc80 complex for assembly into the kinetochore. Further, Ndc80 requires Zwint for assembly, indicating a co-dependency for these two proteins. Our data provide a model for the structural architecture and assembly pathway of the vertebrate kinetochore.     

The three-dimensional architecture of chromosome fibres in the crane fly

Microtubules of two chromosome fibres in metaphase and two in anaphase of the first meiotic division of Pales ferruginea spermatocytes were traced in serial sections. The number and lengths of microtubules constituting the fibres were determined and the fibres reconstructed. The chromosome fibres were found to contain only about 20% (in metaphase) and 9% (in anaphase) kinetochore microtubules (kMT), taking the sum of all individual microtubule lengths as the measure. The remainder consisted of free microtubules (fMT), a certain percentage of which were oriented obliquely with respect to the kMTs (skew fMTs). Between metaphase and anaphase most kMTs and fMTs shortened, causing a decrease in mean length of about onehalf in the case of kMTs. The proportion of skew fMTs increased considerably in anaphase, leading to more disorder in the fibre. The absolute mass of microtubules in the fibre seems to be irrelevant for spindle function. This can be deduced from the observed variability of absolute mass. A statistical correlation was found between the total mass of microtubules (kMTs+fMTs) and the number of kMTs, suggesting that some functional interrelation exists between these parameters.Dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.     

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore-microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.