Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension

In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.     

Isolation and structural organization of human mitotic chromosomes

New methods are presented for the bulk isolation of metaphase chromosomes from HeLa cells, and an electron microscopic study of thin sections of these chromosomes is presented. The techniques for chromosome isolation were developed to utilize solution conditions that are as mild as possible, so that further biochemical and structural studies can be directly related to the in situ state of chromosomes. — Electron micrographs of thin sections of isolated HeLa metaphase chromosomes reveal the general organization of the nucleosome-containing fibers. Chromosomes in isolation buffer show a dense, relatively uniform distribution of material across the chromatids. Swollen chromosomes reveal the primary mode of organization of the fibers to be a radial distribution from the central axes of the chromatids. A significant proportion of the fibers could also be oriented longitudinally.     

DISTRIBUTION OF TRITIUM-LABELED DNA AMONG CHROMOSOMES DURING MEIOSIS : I. Spermatogenesis in the Grasshopper

Thymidine-H³ of high specific activity was used to study the distribution of labeled chromatids during meiotic divisions in spermatocytes of a species of grasshopper (Orthoptera). The distribution is regularly semiconservative as has been shown previously for mitosis, i.e., all chromatids are labeled after incorporation of thymidine-H³ into DNA at premeiotic interphase. If incorporation occurs at the interphase preceding this one, the chromosomes arrive at meiotic divisions with the equivalent of one chromatid of each homologue labeled. Chromatid exchanges occur at a frequency which is very nearly that predicted on the assumption that each chiasma represents an exchange between homologous chromatids. However, the exchanges are randomly distributed among chromosomes in a size group, whereas chiasmata are not. A quantitative analysis of the frequency and pattern of exchanges indicates that most of these result from breakage and reciprocal exchange between homologous chromatids. Sister chromatid exchanges are much less frequent and may be limited to premeiotic stages.     

Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Electron microscopy of unstained BrdU-substituted chromosomes treated with 1.0 M NaH₂PO₄ at high pH and high temperature has demonstrated that there is a structural basis for the light microscopic observation of differentially Giemsa-stained unifilarly and bifilarly BrdU-substituted chromatids and the appearance of chromosome dots. At progressively higher treatment temperatures, sequential structural changes occurred in the chromosomes. After treatment with NaH₂PO₄ at 70–80° C, unifilarly BrdU-substituted chromatids were much more electron opaque than bifilarly substituted chromatids, and the overall data suggest that this difference in electron opacity is a result of the preferential extraction of chromosomal DNA from the bifilarly BrdU-substituted chromatids. NaH₂PO₄ treatment of the BrdU-substituted chromosomes at 80–90° ° C resulted in the formation of highly electron opaque spots (dots) on one or both chromatids. Dots first appeared on the electron lucent bifilarly BrdU-substituted chromatid, indicating that the chromatin with the greatest substitution of BrdU in its DNA is most susceptible to dot formation. At a slightly higher temperature, dots also appeared on the unifilarly BrdU-substituted chromatid concomitant with a disappearance of the electron opacity characterizing this chromatid at the lower treatment temperature. The dots may be formed by an extreme reorganization of residual chromatin or by some kind of interaction or reaction between the chromatin and the salts in the incubation medium. G-band regions may serve as focal points for dot formation.     

A pepsin-revealed material possibly related to chromosomal banding

The enzymes pepsin, alpha-chymotrypsin, trypsin, RNase and DNase were applied to preparations of human metaphase chromosomes before staining to study whether dissociable materials related to the formation of G-, Q- and C-bands would be seen. Treatment with active pepsin but not the other enzymes revealed material with ribonucleo-protein properties which dissociated from the chromosomes and formed a halo.--Lateral extensions from the chromatids stretched to the rim of the halo and appeared at positions corresponding to G-bands. A G-band may be defined as a ring of stable chromatid-matrix binding at positions where the chromatids coil to form lateral extensions.     

Mitomycin C-induced meiotic crossing-over on the interstitial segments in the Chinese hamster heterozygous for a reciprocal translocation

Using Chinese hamsters heterozygous for T(2;10)3Idr and T(1;3)8Idr reciprocal translocations, the authors studied mitomycin C (MMC)-induced crossing-over on the interstitial segments. Marker chromosomes with unequal-length chromatids resulting from crossing-over were clearly detectable, and the frequencies of such marker chromosomes were constant among individual males which were heterozygous for the same reciprocal translocation. The frequency of MMC-induced crossing-over on the interstitial segments increased roughly with increase in dose. These findings, therefore, indicated that marker chromosomes with unequal-length chromatids in translocation heterozygotes may be a useful indicator for detection of the cytogenetic effects of environmental mutagens on germ cells.     

Postmetaphase nuclear formation: Loss of a chromosomal epitope coincident with apparent chromatid coalescence

Previously, we have conceptualized mitotic nuclear formation following metaphase as a morphogenic process and have suggested that sets of chromatids, after separation from a metaphase plate, can be thought of as prenuclei. Such structures can be grouped temporally as either early or late prenuclei based on morphologic, morphometric and density characteristics. Sequential ordering of early prenuclei is of particular interest because it reveals that condensed chromatids coalesce with the resulting formation of a unique chambered structure. In this paper we describe data obtained with a newly raised monoclonal antibody (mAb-2) that initially recognizes an epitope(s) on metaphase chromosomes. Light and confocal fluorescent microscopy of early prenuclei reveal that the chromosomal epitope can no longer be detected about chromatids after their apparent coalescence. Immunoblot analysis of dispersed polypeptides of metaphase plates and early prenuclei indicates that the major protein antigens recognized by mAb-2 have apparent molecular masses of approximately 106000 and 80500 and that each is likely composed of multiple charge isomers. A dual fluorescent analysis using mAb-2 and high-titer anti-lamin B serum provides additional evidence that chromatid coalescence is a separate, early event that precedes nuclear lamina formation. Received: 29 February 1996 / Accepted: 19 March 1996     

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. Here we show that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. Our findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.     

Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.     

Configurational changes in chromatids from helical to banded structures

Induction of configurational changes in the helical chromatids of air dried chromosomes was used to explore the mechanism of G-banding. From the water-Giemsa stained metaphase spreads of Chinese hamster cells, chromosomes having clearly helical chromatids were selected and photographed. Then the chromosomes were decolorized, treated with trypsin, and restained with saline-Giemsa (1 x SSC). Such procedures were repeatedly carried out upon the same chromosomes. Subsequent examination of the chromosomes showed that configurational changes from a helical structure to a banded structure had occurred. Some chromosomes revealed a variety of transitional changes between these two configurations. During the repeated G-banding treatments, the distances between bands along the same chromatids changed each time. The results obtained seem to indicate that the G-banding results from locally induced compaction of chromosomal materials along the chromatids.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.     

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

Scanning electron microscopy of mammalian chromosomes from prophase to telophase

Changes in the morphology of human and murine chromosomes during the different stages of mitosis have been examined by scanning electron microscopy. Two important findings have emerged from this study. The first is that prophase chromosomes do not become split into pairs of chromatids until late prophase or early metaphase. This entails two distinct processes of condensation, the earlier one starting as condensations of chromosomes into chromomeres which then fuse to form a cylindrical body. After this cylindrical body has split in two longitudinally, further condensation occurs by mechanisms that probably include coiling of the chromatids as well as other processes. The second finding is that the centromeric heterochromatin does not split in two at the same time as the rest of the chromosome, but remains undivided until anaphase. It is proposed that the function of centromeric heterochromatin is to hold the chromatids together until anaphase, when they are separated by the concerted action of topoisomerase II acting on numerous similar sites provided by the repetitive nature of the satellite DNA in the heterochromatin. A lower limit to the size of blocks of centromeric heterochromatin is placed by the need for adequate mechanical strength to hold the chromatids together, and a higher limit by the necessity for rapid splitting of the heterochromatin at anaphase. Beyond these limits malsegregation will occur, leading to aneuploidy. Because the centromere remains undivided until anaphase, it cannot undergo the later stage of condensation found in the chromosome arms after separation into chromatids, and therefore the centromere remains as a constriction.by U. Scheer     

Mitosis with undivided chromosomes

Summary Cases of cell division with single chromatids are discussed in connection with a study on mitosis with undivided chromosomes made on living material of the endosperm of Haemanthus katharinae. Such divisions are known from certain abnormal mitoses in the microspores of a few plant species, and also from the second meiotic division, in which it is possible in numerous materials to study the behaviour of daughter univalents, and, in a few cases, also daughter chromosomes derived from chromosomes that were paired during the first division.The various cases of mitosis with single chromatids show a great variation with respect to the degree of scattering of the chromosomes over the spindle at metaphase. In a few cases there is practically no tendency to form a metaphase plate. In other cases the tendency to form such a plate is more or less pronounced, but also in these cases it is difficult for the chromosomes to form this arrangement. Some of them remain scattered over the spindle. After the metaphase a kind of anaphase usually follows in which the single chromatids, without division, move to the poles, often with other chromosomes lagging in intermediate positions.An approach of chromosomes to the poles may be caused by two different mechanisms in mitoses of this kind and only in a few cases is the information sufficient to show that active centromere movements occur during these anaphases.In many aspects of their behaviour on the spindle, single chromatids are similar to ordinary univalents of the first meiotic division. For this reason the movement mechanics of the chromosomes of the first meiotic division is briefly reviewed.The interpretation is expressed that the structure of the centromere region of a single chromatid shows some similarity to that of a univalent of the first meiotic division and that this may be the reason for their similar behaviour. The chromatid centromere would have a structural multiplicity with respect to its kinetic elements, corresponding to its subdivision in half-chromatids and also to the presence of two or three consecutive chromomeres in its longitudinal direction. As these kinetic elements are arranged close to one another on one side of the narrow cylinder of the centromere constriction, it is difficult for them to orient, towards both poles simultaneously. A single chromatid having a centromere of this kind will show orientation instability and change its orientation between the two unipolar orientations and various more or less bipolar orientations. The movements following these different orientations would cause the scattering of these single chromatids over the spindle. The orientation of ordinary mitotic metaphase chromosomes, consisting of two such chromatids, could often be the consequence of a process of co-orientation similar to that in meiotic bivalents.The anaphase movement of undivided chromosomes, which by active centromere movements are shifted in the polar directions without a separation of daughter components, is discussed with reference to a similar behaviour observed by Dietz in multivalents in Ostracods. These multivalents are stabilized in the equator during metaphase, in spite of the fact that they have two or three centromeres directed towards one pole and a single one towards the other. During anaphase their chromosomes do not separate but the whole configurations are shifted towards that pole towards which the majority of the centromeres are directed (this is followed by another type of movement which does not concern us in this connection). Undivided chromosomes that are oriented with more of their kinetic material towards one of the poles and less towards the other should by the same mechanisms as moved the multivalents be shifted in the equatorial direction during metaphase and in the polar direction during anaphase. The mechanism of these events is obscure. A change in the interpretation given by Dietz is suggested.This paper is dedicated to Professor Franz Schrader on the occasion of his seventieth birthday.     

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.     

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

Topographic examination of sister chromatid differential staining by nomarski interference microscopy and scanning electron microscopy

BrdU-substituted Chinese hamster chromosomes were treated with a hot Na₂HPO₄ solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na₂HPO₄ treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.     

Mechanisms of maternal aneuploidy: FISH analysis of oocytes and polar bodies in patients undergoing assisted conception

We have examined unfertilised oocytes and their first polar bodies (PBs) to determine the way in which the frequency of whole chromosome imbalance compares with that involving single chromatids and whether the precocious separation of chromatids prior to anaphase I affects all pairs of chromosomes. We have applied the technique of fluorescent in situ hybridisation in a three-stage method by using locus-specific probes for chromosomes 13 and 21 and alpha-satellite probes for chromosomes 1, 9, 16, 18 and X to determine the chromosome status of oocytes and their PBs. We obtained analysable results from 127 oocytes and 57 PBs from 72 patients of average age 33 years. Six oocytes and three PBs had extra signals but, of these, three were derived from a single patient, aged 26. Anomalies were seen in chromosomes 13, 16, 18, X and, notably, 21 but none were observed in chromosomes 1 and 9. Half of the anomalies involved additional chromatids rather than whole chromosomes. Since particular chromatids were found to be prematurely separated in the metaphase II oocyte, this may provide further evidence for an additional mechanism of maternal aneuploidy that operates at anaphase II. Detailed analyses of both oocytes and PBs have elucidated possible mechanisms leading to aneuploid gametes in this group of patients with fertility problems.