Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 m thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 g/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.     

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.     

The mitotic apparatus in the dinoflagellateAmphidinium carterae

Summary Aspects of mitosis in the dinoflagellateAmphidinium carterae have been examined using TEM, SEM and fluorescence immunochemistry. The extranuclear spindle is composed of 2–4 bundles of microtubules arranged into two interdigitated half-spindles. Unlike previous reports of dinomitosis, the spindle bundles converge at the poles. These bundles of microtubules are inserted into a multilobed, vesiculate body containing electron opaque, amorphous material. This spindle pole body has ribosomes associated with it and is continuous with the endoplasmic reticulum. Chromosomes are attached to the nuclear envelope, which is persistent throughout mitosis. Kinetochore microtubules attach to the nuclear envelope via elongate electron dense kinetochores (one microtubule per daughter kinetochore). Several microtubules pass alongside the kinetochore, forming a halo of 3–4 spindle microtubules. Electron dense connections can be seen between some of these microtubules and the kinetochore. Chromosome segregation appears to be a function of spindle elongation (anaphase B), since chromosome-to-pole distance (anaphase A) remains relatively unchanged throughout mitosis.Abbreviations DABCO 1,4 diazabicyclo(2,2,2)octane - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,NN-tetraacetic acid - PIPES piperazine-N,N-bis(2-thanesulfonic acid) Supported by a Charles and Johanna Bush Predoctoral Fellowship to S. B. B.     

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 and microtubules in two members of Chlorophyceae,Cladophora fracta and Spirogyra majuscula

Evidence is presented for the existence of a localised kinetochore with stratified fine structure in Cladophora and in Spirogyra. In the latter, there is the possibility of two kinetochores on the longer chromosomes. There is no evidence for a diffuse kinetochore. The nucleolus persists during mitosis in Cladophora on the nucleolar organising chromosomes, the granular material being lost from it very largely during metaphase and anaphase but the fibrillar material remaining. The persistent nucleolar material at metaphase and anaphase in Spirogyra is not attached to the nucleolar organising chromosomes but accumulates around all the chromosomes and chromatids, the microtubules of the spindle at anaphase passing through and possibly attaching to this nucleolar material and possibly assisting in the movement of the chromatids which are embedded within it.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.     

Formation of spindle fibers,kinetochore orientation,and behavior of the nuclear envelope during mitosis in endosperm

The formation of kinetochore (chromosomal) and continuous fibers, and the behavior of the nuclear envelope (NE) was described in studies combining light and electron microscopy. Microtubules (MTs) push and pull the NE which becomes progressively weaker before breaking. It breaks to a certain extent due to mechanical pressure. Clear zone MTs penetrate into the nuclear area as dense bundles and form continuous fibers. These MTs also attach to some kinetochores during this process. Some kinetochore fibers seem to be formed by the kinetochores themselves which are also responsible for further development and changes of kinetochore fibers. Formation of kinetochore fibers is asynchronous for different chromosomes and even for two sister kinetochores. Often temporary faulty connections between different kinetochores or the polar regions are formed which usually break in later stages. This results in movements of chromosomes toward the poles and across the spindle during prometaphase. The NE, whose fine structure has been described, breaks into small pieces which often persist to the next mitosis. Old pieces of NE are utilized in the formation of new NE at telophase. Several problems concerning the mechanism of chromosome movements, visibility of the NE, etc., have also been discussed.     

Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301–1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242–246; Li, Y., and R. Benezra. Science. 274: 246–248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK₁ cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929–939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK₁ cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after ~12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.     

The kinetochores of Caenorhabditis elegans

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 m in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18–0.2 m in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 m, and an electron lucent middle layer of 0.03 m. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.     

Nuclear division in the cyst,and white spot nuclei preceding cyst formation,inAcetabularia wettsteinii

Summary Electron microscopy of nuclear division in young cysts ofAcetabularia wettsteinii shows that the dividing nucleus hat two additional cisternae of endoplasmic reticulum immediately outside the nuclear envelope. These additional cisternae are attached to, and apparently formed from a membrane body which develops outside the nucleus in early prophase. The interphase nucleus does not have the additional cisternae. The nucleoli are extruded from the nucleus at anaphase, the nucleolar bodies remaining in the peri-nuclear cytoplasm. The chromosomes have localized centromeres; the stratified ultrastructure characteristic of some chlorophycean and animal kinetochores has not been found inAcetabularia, although the kinetochore appears distinct, projecting from the chromatid, and has attached microtubules. The condensed bodies of the white spot nucleus are discussed.     

Acetylation of α-tubulin in male meiotic spindles ofPyrrhocoris apterus,an insect with holokinetic chromosomes

Summary Kinetochore structure was examined in metaphase spermatogonia and primary spermatocytes of the red firebug,Pyrrhocoris apterus (Pyrrhocoridae, Hemiptera). Chromosome spreads were analysed using light microscopy and serial sections through spindles were studied using electron microscopy. Mitotic chromosomes were rod-shaped bodies and did not possess primary constrictions. Trilaminar kinetochores occurred throughout about 72% of the chromosomal length. Numerous microtubules (MTs) were connected with the outer plates of the kinetochores and interactions between MTs and the remainder of the chromosomal surface were rare. The bivalents formed dumbbell-shaped bodies in metaphase I spermatocytes. At that stage, MTs were found in contact with the entire poleward surface of the chromosomes. Distinct kinetochore material was, however, not detectable and some MTs penetrated deeply into the chromatin. Mitotic and meiotic chromosomes ofP. apterus are holokinetic and consequently the number of kinetochore MTs is expected to be relatively high. In the second part of the study, the question whether holokinetic chromosomes affect spindle MT dynamics is addressed. To this end, primary spermatocytes ofP. apterus were labelled with a widely used antibody, 6-11B-1, directed against acetylated -tubulin. The acetylation of -tubulin is believed to indicate the presence of long-lived MTs. MT bundles were labelled in metaphase and anaphase I spindles, while prophase and prometaphase I spermatocytes did not contain acetylated MTs. MTs in early and mid telophase spindles were not acetylated. Only late telophase I spindles possessed small amounts of acetylated -tubulin. The acetylated MT bundles of metaphase and anaphase I spindles probably represent kinetochore MTs stabilized by their association with the holokinetic chromosomes at one end and the spindle poles at the opposite end.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole · 2HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis(2-ethane sulfonic acid) - MT microtubule     

Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 m sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.     

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

Background

Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.

Principal Findings

We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells.

Conclusions/Significance

Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.     

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.     

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.     

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada)

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.     

Cytochemistry of kinetochores under electron microscopy

A cytochemical analysis has been performed on kinetochores of mouse, Allium and grasshopper under the electron microscope. The study was carried out using serial sections and cytochemical methods. Alcoholic PTA was used for basic protein staining and the EDTA method for preferential staining of ribonucleoproteins. In mouse and Allium chromosomes the kinetochore appears positively stained after PTA and EDTA. In grasshopper chromosomes, kinetochores appear as a fibrillar and less dense region and are positively stained after EDTA. Blocks from mouse treated with HCl prior to PTA stain show lower contrast in the kinetochore. When Allium cepa anthers were treated with RNase and perchloric acid (PCA) there was no positive effect after EDTA stain in the kinetochore region. It is suggested that non-DNA material takes part in the constitution of the kinetochore. This material would be made up, at least in part, of basic proteins and ribonucleoproteins.     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

Meiosis in Drosophila melanogaster

Individual bivalents or chromosomes have been identified in Drosophila melanogaster spermatocytes at metaphase I, anaphase I, metaphase II and anaphase II in electron micrographs of serial sections. Identification was based on a combination of chromosome volume analysis, bivalent topology, and kinetochore position. — Kinetochore microtubule numbers have been obtained for the identified chromosomes at all four meiotic stages. Average numbers in D. melanogaster are relatively low compared to reported numbers of other higher eukaryotes. There are no differences in kinetochore microtubule numbers within a stage despite a large (approximately tenfold) difference in chromosome volume between the largest and the smallest chromosome. A comparison between the two meiotic metaphases (metaphase I and metaphase II) reveals that metaphase I kinetochores possess twice as many microtubules as metaphase II kinetochores. — Other microtubules in addition to those that end on or penetrate the kinetochore are found in the vicinity of the kinetochore. These microtubules penetrate the chromosome rather than the kinetochore proper and are more numerous at metaphase I than at the other division stages.