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     

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.     

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.     

Kinetochore structure: electron spectroscopic imaging of the kinetochore

The structure of the kinetochore in thin section has been studied in the Indian muntjac by an electron spectroscopic imaging technique. This procedures allows the analysis of the distribution of phosphorus within the layers of the kinetochore. The results indicate that this element is a major component of both the inner and outer plates whereas it is largely absent in the middle plate and fibrous corona. The majority of the phosphorus is localized to a 30-nm fiber(s) that is woven through the layers of the kinetochore. The presence of phosphorus within this fiber, along with its morphological and biochemical features, indicates that it contains DNA. The fiber(s) occupies a major portion of the inner and outer plate where it forms a series of rows. It is rarely observed in the middle layer except where it passes between the inner and outer layers. The absence of structure in the middle plate suggests that it may represent a space rather than a plate that in turn may be related to the function of this region. The distribution of phosphorus within the kinetochore is neither altered by treatment with colcemid nor by the presence of microtubules at the kinetochore. Analysis of conventional micrographs of the kinetochore together with structural information obtained by electron spectroscopic imaging suggests that most microtubules insert and terminate between the rows of kinetochore fibers in the outer plate. However, some microtubules continue through the middle layer and terminate at the lower plate. The insertion of microtubules at different levels of the kinetochore may reflect the existence of functionally distinct microtubule classes. Electron spectroscopic imaging indicates that the microtubules associated with the kinetochore are phosphorylated.     

The organization of the mammalian kinetochore: a scanning electron microscope study

Summary A procedure has been developed for scanning electron microscopy that enables the visualization of kinetochores along the surface of isolated chromosomes of the Indian muntjac. Indirect immunofluorescence and thin section analysis of the kinetochores of those isolated chromosomes verified that these structures retained in vivo composition and morphology during the isolation procedure. In scanning electron micrographs the outer surface of the outer kinetochore plate can be visualized as a series of fibers 25–30 nm in diameter that are arranged across the plate. These images are comparable to those obtained by whole mount transmission electron microscopic procedures (Rattner 1986) and are compatible with a model of the kinetochore in which chromatin fiber from the body of the chromosome extend to the outer kinetochore plate.     

Organization within the mammalian kinetochore

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25–30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.     

The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.     

Structure of the colcemid-treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic tomography

High voltage electron microscopic tomography was used to determine the organization of the kinetochore plate and its attachment to the underlying chromosome. Six reconstructions were computed from thick sections of Colcemid-treated PtK1 cells and analyzed by a number of computer graphics methods including extensive thin slicing, three- dimensional masking, and volume rendering. When viewed en-face the kinetochore plate appeared to be constructed from a scaffold of numerous 10-20-nm thick fibers or rods. Although the fibers exhibited regions of parallel alignment and hints of a lattice, they were highly variable in length, orientation and spacing. When viewed in stereo, groups of these fibers were often seen oriented in different directions at different depths to give an overall matted appearance to the structure. When viewed "on edge," the plate was 35-40 nm thick, and in thin slices many regions were tripartite with electron-opaque domains, separated by a more translucent middle layer, forming the inner and outer plate boundaries. These domains were joined at irregular intervals. In some slices, each domain appeared as a linear array of 10- 20-nm dots or rods embedded in a less electron-opaque matrix, and adjacent dots within or between domains often appeared fused to form larger blocks. The plate was connected to the underlying chromosome by less densely arrayed 10-20-nm thick fibers that contacted the chromosome-facing (i.e., inner) surface of the plate in numerous patches. These patches tended to be arrayed in parallel rows perpendicular to the long axis of the chromosome. In contrast to connecting fibers, corona fibers were more uniformly distributed over the cytoplasmic-facing (i.e., outer) surface of the plate. When large portions of the reconstructions were viewed, either en-face or in successive slices parallel to the long axis of the chromosome, the edges of the plate appeared splayed into multiple "fingers" that partly encircled the primary constriction. Together these observations reveal that regions of the kinetochore outer plate contain separate structural domains, which we hypothesize to serve separate functional roles. Our three-dimensional images of the kinetochore are largely consistent with the hypothesis that the outer plate is composed of multiple identical subunits (Zinkowski, R. P., J. Meyne, and B. R. Brinkley. 1991. J. Cell Biol. 113:1091-1110).     

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4–6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users.     

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.     

Structure of the mammalian kinetochore

The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D₂O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer. We dedicate this paper to Wolfgang Beermann on the occasion of his 60th birthday in appreciation of many years of friendship and his pioneering contributions in the field of chromosome biology     

Dynamic distribution of TTK in HeLa cells： insights from an ultrastructural study

Entry into mitosis is driven by signaling cascades of mitotic kinases. Our recent studies show that TTK, a kinetochore-associated protein kinase, interacts with CENP-E, a mitotic kinesin located to corona fiber of kinetochore. Using immunoelectron microscopy, here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells. Upon nuclear envelope fragmentation, TTK targets to the outermost region of the developing kinetochores of monoorient chromosome as well as to spindle poles. After stable attachment, throughout chromosome congression, TTK is a constituent of the corona fibers, extending up to 90 nm away from the kinetochore outer plate. Upon metaphase alignment, TTK departs from the kinetochore and migrates toward the centrosomes. Taken together, this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.     

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     

CENP-B: a major human centromere protein located beneath the kinetochore

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.     

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.     

Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.     

Mapping DNA within the mammalian kinetochore

The location of the cis-acting DNA sequences that direct the assembly of the mammalian kinetochore is not known. A variety of circumstantial evidence, however, has led to the widespread belief that they are present throughout the kinetochore including the kinetochore outer plate. To investigate this question directly, we have used two independent methods to localize DNA in and around the mammalian kinetochore. Both methods fail to reveal DNA in the outer kinetochore plate, finding instead that the outer-most detectable DNA in the centromere is located in the inner kinetochore plate. Our results imply that the outer kinetochore plate is primarily a proteinaceous structure. It is thus unlikely that fibers observed in the outer plate correspond to chromatin, as previously assumed. Our observations suggest that current models of kinetochore structure may need to be reconsidered.     

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways. Received: 6 August 1998 / Accepted: 28 August 1998     

Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.