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.     

Analysis of detached human kinetochores

A method has recently been established for inducing the physical detachment of kinetochores from chromosomes in human HeLa cells, and was used in the studies reported here to investigate the organization and function of dissociated HeLa kinetochores. Immunofluorescence labeling demonstrated that the detached HeLa kinetochores were relatively intact, with the number of detached kinetochores being only moderately more than the diploid number of chromosomes in HeLa cells. In addition, the detached kinetochores could be labeled with antibodies specific for the inner kinetochore plate, outer kinetochore, and subjacent centromeric heterochromatin. A functional assay demonstrated that detached kinetochores retained the capacity to activate the spindle checkpoint, leading to metaphase arrest. Analysis of kinetochore DNA indicated that it consisted primarily of DNA fragments of 130–160 kb in size, while the remainder of the chromosomes were sheared into much smaller fragments during the kinetochore detachment event. Further analysis of kinetochore DNA indicated that it was first cleaved into high molecular weight DNA (>200 kb) fragments during the initial stages of the kinetochore detachment process, and then underwent further maturation following nuclear envelope breakdown to give rise to the 130–160 kb fragment in detached kinetochores. Collectively, these data indicate that detached human kinetochores will be a useful system for investigating the organization, assembly, and function of human kinetochores. Edited by: W.C. Earnshaw     

The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis

The kinetochore binds an evolutionarily conserved set of checkpoint proteins that function to monitor whether chromosomes have aligned properly at the spindle equator. Human cells contain two related protein kinases, hBUB1 and hBUBR1, that appear to have evolved from a single ancestral BUB1 gene. We generated hBUB1- and hBUBR1-specific antibodies so that the localization patterns of these kinases could be directly compared. In the human U2OS osteosarcoma cell line, hBUB1 first appeared at kinetochores during early prophase before all kinetochores were occupied by hBUBR1 or CENP-F. Both proteins remained at kinetochores throughout mitosis but their staining intensity was reduced from anaphase onward. Kinetochores of unaligned chromosomes exhibited stronger hBUB1 and hBUBR1 staining. Immunoelectron microscopy showed that hBUBR1 appeared to be concentrated in the outer kinetochore plate and in some instances the inner plate as well. When chromosome spreads were examined by light microscopy, hBUB1 and hBUBR1 were coincident with CENP-E. This suggests that both kinases are concentrated near the surface of the kinetochore where they can monitor kinetochore-microtubule interactions. Received: 8 August 1998 / Accepted: 13 September 1998     

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.     

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

Light and electron microscopy of rat kangaroo cells in mitosis

Kinetochores in rat kangaroo (PtK₂) cells in prophase of mitosis are finely fibrillar, globular bodies, 5000–8000 Å in diameter. Sister kinetochores are attached to opposite lateral faces in the primary constriction of chromosomes. No microtubules (MTs) occur in prophase nuclei. During prometaphase the ball-shaped kinetochores differentiate into trilaminar plaques. An outer kinetochore layer, less electron dense than chromatin, appears first in the fibrillar matrix. The inner layer, continuous with, but more electron dense than the chromosome, is formed later. Kinetochore-spindle MT interaction is evident at the very beginning of prometaphase. As a result, kinetochore shape is very variable, but three types of kinetochores can be distinguished by fine structure analysis. A comparison of kinetochore structure and chromosome position in the mitotic spindle yielded clues regarding initial orientation and congression. At the time the nuclear envelope (NE) breaks down chromosomes near asters orient first. Chromosomes approximately equidistant from the two spindle poles amphi-orient immediately. Chromosomes closer to one pole probably achieve mono-orientation first, then amphi-orient and congress. In normal metaphase all the chromosomes lie at or near the spindle equator and kinetochores are structurally uniform. Paraxial and para-equatorial sections revealed that they are trilaminar, roughly circular plaques of 4000–6000 Å diameter. Inner and outer layers are 400 Å, and the electron translucent middle layer which separates them is 270 Å thick. From 16 to 40 MTs are anchored in the outer layer. In cold-treated cells the kinetochores are trilaminar, but in colcemid-treated cells the inner layer is lacking. Both kinetochores and their MTs are disorganized beginning in late anaphase. In telophase the inner layer persists for some time as an electron dense patch apposed to the NE, while the outer layer disintegrates.     

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.     

Holokinetic composite chromosomes with “diffuse” kinetochores in the micronuclear mitosis of a heterotrichous ciliate

During micronuclear mitosis of the heterotrichous ciliate Nyctotherus ovalis Leidy rod-shaped composite chromosomes are formed by lateral association of telokinetic chromosomes. The formation of these composite chromosomes seems to be a highly ordered process since only nuclei with either 18 or 24 such chromosomes can be observed, and nuclei with the same chromosome number show a similar length distribution of their chromosomes. Further, these data indicate that we examined two otherwise indistinguishable races. During metaphase the composite chromosomes become arranged in the spindle equator in a holokinetic fashion, their entire poleward surfaces being covered by kinetochore material. These diffuse kinetochores have a trilaminar appearance comparable to those of monokinetic chromosomes. Their electron density after employing Bernhard's procedure revealed the same ribonucleoprotein distribution as reported for the localized kinetochores formed during the extranuclear mitosis in other cells. During early anaphase the outer kinetochore layer remains continuous while the individual chromosomes in the composite group show a tendency to separate leaving chromatin-free spaces of about 40 nm diameter. Kinetochore microtubules which are still anchored in the outer kinetochore layer seem to elongate and to extend into the interpolar spindle region predominantly through these holes in the chromatin. These observations suggest a like polarity of kinetochore and interpolar microtubules in the polar spindle region while microtubules in the interpolar space seem to interdigitate in an antiparallel fashion. The activity of the kinetochore to act as a microtubule-organizing center (MTOC) seems to be modulated by the chromatin underlying the outer kinetochore layer which may prevent further outgrowth of kinetochore microtubules.     

Mitosin／CENP-F is a conserved kinetochore protein subjected to cyto-plasmic dynein-mediated poleward transport

Mitosin/CENP-F is a human nuclear protein transiently associated with the outer kinetochore plate in M phase and is involved in M phase progression. LEK1 and CMF1, which are its murine and chicken orthologs, however, are implicated in muscle differentiation and reportedly not distributed at kinetochores.We therefore conducted several assays to clarify this issue. The typical centromere staining patterns were observed in mitotic cells from both human primary culture and murine, canine, and mink cell lines. A C-terminal portion of LEK1 also conferred centromere localization. Our analysis further suggests conserved kinetochore localization of mammalian mitosin orthologs. Moreover, mitosin was associated preferentially with kinetochores of unaligned chromosomes. It was also constantly transported from kinetochores to spindle poles by cytoplasmic dynein. These properties resemble those of other kinetochore proteins important for the spindle checkpoint, thus implying a role of mitosin in this checkpoint. Therefore, mitosin family may serve as multifunctional proteins involved in both mitosis and differentiation.     

Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.     

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electron-translucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK₁) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50–75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100–150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures. Received: 7 August 1998; in revised form: 18 August 1998 / Accepted: 20 August 1998     

Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase

We have conducted a detailed ultrastructural analysis of the distribution of the kinesin-related centromere protein CENP-E during mitosis in cultured human, rat kangaroo and Indian muntjac cells. Using an affinity-purified polyclonal antibody and detection by 0.8 nm colloidal gold particles, CENP-E was localized primarily to the fibrous corona of the kinetochore in prometaphase and metaphase cells. Some labeling of the kinetochore outer plate was also observed. The distribution of fibrous corona-associated CENP-E did not change dramatically following the attachment of microtubules to the kinetochore. Thus, the normal disappearance of this kinetochore substructure in conventional electron micrographs of mitotic chromosomes with attached kinetochores is not due to the corona becoming stretched along the spindle microtubules as has been suggested. Examination of cells undergoing anaphase chromatid movement revealed the presence of CENP-E still associated with the outer surface of the kinetochore plate. At the same time, the majority of detectable CENP-E in these cells was associated with the bundles of antiparallel microtubules in the central spindle. CENP-E in this region of the cell is apparently associated with the stem body matrix material. The simultaneous localization of CENP-E on centromeres and the central spindle during anaphase was confirmed by both wide-field microscopy of human cells and conventional fluorescence microscopy of rat kangaroo cells. Together, the observations reported here are consistent with models in which CENP-E has a role in promoting the poleward migration of sister chromatids during anaphase A. Received: 21 July 1997 /Accepted: 19 September 1997     

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.     

Silencing mitosin induces misaligned chromosomes, premature chromosome decondensation before anaphase onset, and mitotic cell death

Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.     

HIM-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes

Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.     

Holocentric chromosomes in Oncopeltus: kinetochore plates are present in mitosis but absent in meiosis

Fine structure studies of Oncopeltus fasciatus, an hemipteran with diffuse kinetochores, shows the presence of a kinetochore plate extending for up to 75% of the length of the chromosomes during mitosis. During meiosis, microtubules entered all along the body of the chromosomes and the kinetochore plate was completely missing. It is suggested that in organisms with holocentric chromosomes the formation of the meiotic kinetochore apparatus may have to be suppressed to allow terminalization of chiasmata.Supported by N.I.H. Grant No. GM-15886.     

Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase

Prometaphase in two large species of diatoms is examined, using the following techniques: (a) time-lapse cinematography of chromosome movements in vivo; (b) electron microscopy of corresponding stages: (c) reconstruction of the microtubules (MTs) in the kinetochore fiber of chromosomes attached to the spindle. In vivo, the chromosomes independently commence oscillations back and forth to one pole. The kinetochore is usually at the leading edge of such chromosome movements; a variable time later both kinetochores undergo such oscillations but toward opposite poles and soon stretch poleward to establish stable bipolar attachment. Electron microscopy of early prometaphase shows that the kinetochores usually laterally associate with MTs that have one end attached to the spindle pole. At late prometaphase, most chromosomes are fully attached to the spindle, but the kinetochores on unattached chromosomes are bare of MTs. Reconstruction of the kinetochore fiber demonstrates that most of its MTs (96%) extend past the kinetochore and are thus apparently not nucleated there. At least one MT terminates at each kinetochore analyzed. Our interpretation is that the conventional view of kinetochore function cannot apply to diatoms. The kinetochore fiber in diatoms appears to be primarily composed of MTs from the poles, in contrast to the conventional view that many MTs of the kinetochore fiber are nucleated by the kinetochore. Similarly, chromosomes appear to initially orient their kinetochores to opposite poles by moving along MTs attached to the poles, instead of orientation effected by kinetochore MTs laterally associating with other MTs in the spindle. The function of the kinetochore in diatoms and other cell types is discussed.     

A Highlights from MBoC Selection: Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.