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.     

Vertebrate kinetochore protein architecture: protein copy number

To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores.     

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.     

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.     

Measuring nanometer scale gradients in spindle microtubule dynamics using model convolution microscopy

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.     

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.     

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     

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 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.     

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.     

Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.     

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.     

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.     

CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore‐microtubule dynamics to promote mitotic progression and fidelity

Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore–microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b–CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin–CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1–astrin–Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.     

Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.     

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this limit, which may explain why it has the smallest known mitotic spindle that still manifests the classic congression architecture.     

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.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.