Structure of the human centromere at metaphase

Until recently the centromere was thought to be a relatively homogeneous region of densely packed heterochromatin with a single differentiated domain--the kinetochore--at its surface, representing the point of attachment of the mitotic spindle. We now know that the centromere of higher eukaryotes is composed of several domains that have been identified using antibody probes. Somewhere within the domains are located both the factor(s) that control the disjunction of sister chromatids and the molecular motor responsible for chromosome movement towards the spindle poles.     

Patterns of association in the human metaphase complement: Ring analysis and estimation of associativity of specific chromosome regions

Summary The pattern of metaphase chromosome association in the human complement was studied by two methods of statistical analysis of interchromosomal distances. Those methods included ring analysis in which a characteristic position of the centromere of each chromosome relative to the center of a two-dimensional representation of a metaphase complement was defined, and estimation of the capacity for associativity of each of three regions of each chromosome: the centromere (c) and the ends of each arm (p,q).The following information was obtained: 1. In general, the distance from the center is directly related to chromosome size. 2. The most notable deviation from that size-related progression is displayed by the X chromosomes. The markedly peripheral position of the X is characteristic of both X's of the female and the single X of the male. 3. The relative associativity of each chromosome of the complement is, in general, inversely related to size with an additional preferential capacity of associativity displayed by the acrocentric chromosomes. Analyses of the different inter-regional classes established that the supplementary associativity factor of the acrocentric chromosomes was inherent in their pericentromeric and p-arm regions and excluded the ends of the q arms from participation in that factor. 4. Those analyses demonstrated that the specific morphology or geometry of the acrocentric chromosomes contributes little to their high relative associativity. In addition to the tendency for the c/p regions of the acrocentric chromosomes to associate with each other, presumably because of their common function in nucleolar organization, those regions also displayed a propensity to associate with the distal regions of the arms of other chromosomes. A molecular basis for that propensity other than that of ribosomal DNA is postulated to be that of other fractions of highly reiterated DNA sequences. 5. Analysis of the relative associativities of each of the three regions of the Y chromosome revealed that the Yq displays a much stronger capacity to associate with the c's of other chromosomes than does the Yc or Yp.     

Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.     

Alignment of BrdU-substituted chromatids in satellite association in human metaphase cells

We have studied the orientation of BrdU-substituted chromatids in satellite associations in cells double-stained to reveal both the Ag/As nucleolar organizer regions and, simultaneously, sister chromatid differentiation. In those satellite associations with all four chromatids joined by Ag stain, and with the axes in a virtually straight line, we have observed an excess of concordant configurations. Where an association was considered single, dark chromatids were involved in significantly more associations than light chromatids. Within this group, the observed excess of concordant associations was not greatly different from the numbers observed in the straight, double-chromatid group of configurations. Whether the increased involvement of dark chromatids in satellite associations provides a complete explanation for the observed excess of concordant associations, or whether certain individuals show a specific tendency to form concordant associations, must await further data.     

Telomere association of human chromosomes induced by aphidicolin.

Telomere associations were studied in metaphase chromosomes from 96-h cultures of peripheral blood lymphocytes of two healthy women, treated with 0.4 microM aphidicolin for the last 72 h. Telomere associations were encountered in 2.9% and 3.2% of the metaphases screened, whereas no such associations were encountered in 5-fluorodeoxyuridine-treated cultures. The chromosome arms involved in telomere associations were nonrandom: 1q, 2q, 3q, 6p and 16q were more frequently involved in the associations (P less than 0.01). Of the 51 combinations of telomere associations encountered, those occurring nonrandomly were 1q/2q, 2q/2q, 4q/4q, 6q/6q and 6p/6p associations.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Correlation between centromere and chromosome length in human male pronuclear chromosomes: ultrastructural analysis

Ultrastructural and morphometric analyses of centromeric regions by scanning and transmission electron microscopy have been performed in chromosomes from male pronuclei obtained by heterologous fertilisation of hamster oocytes with human spermatozoa. In 1308 of 1323 chromosomes analysed, the primary constriction showed a defined biconcave constriction of variable length (0.56-1.34 microns) and constant width (0.64-0.7 micron). A positive correlation was observed between centromeric length and chromosome length. In some chromosomes, the primary constriction appears as decondensed regions of variable length (1.6-2.51 microns) composed of chromatin fibres with a minimum diameter of 30 nm.     

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.     

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.     

Telomere dynamics in human cells

Human telomeres are intrinsically dynamic structures, with multiple biological processes operating to generate substantial length heterogeneity. Processes that operate specifically at the terminus, that include the end-replication problem coupled with C-strand resection, result in gradual telomere erosion with ongoing cell division. Rates of telomere erosion can be modulated by cell culture conditions and pleiotropic effects. Other processes, that are not consistent with the end replication problem, generate sporadic large-scale changes in telomere length. These events are detected in normal human cells and tissues; the severely truncated telomeres that result are potentially fusogenic and may lead to the types of genetic rearrangements that typify early-stage neoplasia. The processes that underlie sporadic telomeric deletion are unclear, but may include intra-allelic recombination within the T-loop structure, unequal sister chromatid exchange and replication fork stalling. The relative contributions of these processes in the generation of the heterogeneous telomere length profiles observed in human cells are discussed.     

The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.     

Telomere and telomerase dynamics in human cells

Accumulating evidence now implicates telomeres and telomerase as critical regulators genomic stability and replicative lifespan in mammalian cells. Disruption of telomere maintenance and/or telomerase expression contributes to the etiology of some degenerative diseases and may participate in the process of aging. Although telomere dysfunction and aberrant telomerase expression clearly play important roles in cancer development, the contribution of telomere biology to cancer is complex and involves both positive and negative influences on tumor development. Indeed, recent work from several laboratories suggests additional roles for telomeres and telomerase in both normal and malignant physiology. Understanding the complexity of telomere biology will provide further insights into chromosome biology in both normal and malignant cells.     

On the cell-free association of lamins A and C with metaphase chromosomes

Nuclear envelopes have previously been shown to assemble spontaneously around endogenous chromosomes in cell-free homogenates of mitotic Chinese hamster ovary cells. In order to further analyze the mechanisms underlying nuclear envelope reformation and the functions of the individual nuclear lamin polypeptides, a fractionated cell-free nuclear envelope reassembly system involving purified chromosomes and either a postchromosomal supernatant or a cytosol fraction from mitotic cells has been devised. Results obtained with this fractionated system show that lamins A and C will associate with the surfaces of chromosomes in the absence of lamin B and membranes, this association being inhibitable by ATP-gamma-S. However, in the absence of membranes chromatin decondensation never occurs. Using the reversible swelling of chromosomes in low ionic strength buffers lacking divalent cations as the basis of a simple assay, it is demonstrated that the association of lamins A and C with the surfaces of chromosomes has a pronounced and easily observable effect on chromatin organization.     

Analysis of metaphase I chromosome association in species of the genus Aegilops

Summary Metaphase-I chromosome associations in every diploid and polyploid species of the genus Aegilops were studied using C-banding in order to analyse the cytogenetic behaviour of the whole complement as well as of specific genomes in different polyploid species. Differences were observed in the frequency of associations per cell among different species of the same ploidic level and even between species sharing the same genomic constitution. Differences were also found between different genomes within the same polyploid species and between the same genome when present in several diploid and polyploid species. Several factors proposed as having an influence on the frequency of metaphase-I associations, such as chromosome morphology, C-heterochromatin content, genetic control and genome interactions, are discussed. Most of the polyploid Aegilops species showed a diploid-like behaviour at metaphase I although multivalents involving homoeologous associations were occasionally observed in Ae. biuncialis, Ae. juvenalis and Ae. crassa(6x); therefore, the Aegilops diploidising genetic system is not equally effective in all polyploid species.     

Effects of vitrification for germinal vesicle and metaphase II oocytes on subsequent centromere cohesion and chromosome aneuploidy in mice

The present study examined the effect of vitrification on oocyte aneuploidy and centromere cohesion. Firstly, germinal vesicle (GV) and in vitro matured oocytes (metaphase II, MII) were vitrified by open-pulled straw method. Secondly, thawed GV oocytes were matured in vitro to detect the aneuploidy rate and the sister inter-kinetochore (iKT) distance (in situ spreading and immunofluorescent staining). The results revealed that the sister iKT distance and the aneuploidy rate in eggs matured from vitrified-thawed GV oocytes were higher than that from in vivo matured, in vitro matured, and in vitro matured frozen oocytes (0.47 ± 0.03 vs. 0.33 ± 0.01 vs. 0.33 ± 0.02 vs. 0.34 ± 0.01 μm; P < 0.01 and 22.9% vs. 6.5% vs. 5.8% vs. 11.8%; P < 0.05, respectively). Furthermore, the percentage of sister chromosome pairs whose sister iKT distances were higher than 0.9 μm in eggs matured from vitrified-thawed GV oocytes (8.7%) was higher than that from in vivo matured (1.6%), in vitro matured (1.6%), and in vitro matured frozen oocytes (2.3%) (P < 0.05). The sister iKT distance was associated with centromere cohesion. To investigate whether vitrification of GV oocytes deteriorated centromere cohesion by affecting cohesin complex formation, thawed and fresh GV oocytes were used to detect the cohesin subunits (SMC1β, STAG3, SMC3, and REC8) mRNA expression (quantitative real-time polymerase chain reaction). The relative expression of three cohesin subunits (SMC1β, STAG3, and SMC3) was significantly decreased in GV oocytes after vitrification. In conclusion, vitrification of GV oocytes may result in the subsequent deterioration of centromere cohesion and an increase in the aneuploidy rate. MII oocytes may be the ideal candidate to avoid aneuploidy for fertility cryopreservation.     

HCP-4/CENP-C promotes the prophase timing of centromere resolution by enabling the centromere association of HCP-6 in Caenorhabditis elegans

Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.     

Banding and spiralization of human metaphase chromosomes

Summary A new technique is described which produces spiralization of human metaphase chromosomes. The important feature is heat followed by trypsin treatment. By varying conditions, it is possible to produce bands, spirals and intermediate stages. This provides a new approach to the understanding of banding and chromosome structure.