The three-dimensional architecture of chromosome fibres in the crane fly

Microtubules of amphitelically oriented sex univalent chromosome fibres were traced in longitudinal serial sections. The investigated chromosomes were from four different cells representing consecutive stages of anaphase segregation. A correlation was found between chromosome movement and a characteristic distribution of free microtubules (fMTs) oriented obliquely with respect to the kinetochore microtubules. During chromosome segregation the proportion of these skew fMTs (the proportion of skew fMTs is a measure of the degree of disorder in the fibre) is higher in the fibre pointing in the direction of movement than in the trailing fibre. The results are discussed in relation to spindle forces. Although the anaphase of amphitelic sex chromosomes is different in several respects (orientation of chromosome fibres, mutual connexion of chromosomes via kinetochore microtubules, spindle elongation occurring simultaneously), the observations on the distribution of fMTs in the chromosome fibres is, in principle, compatible with those previously made on syntelic autosomes.     

Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.     

Stable versus unstable orientations of sex chromosomes in two grasshopper species

The structural basis of orientation stability was investigated. The stable unipolar orientation of the Melanoplus sanguinipes X-chromosome univalent is unique in that it is stable without tension created by forces towards opposite poles; tension is thought to be the principle component in stabilizing kinetochore orientations to a pole. Stable orientation of the X chromosome in Melanoplus sanguinipes was compared with unstable X orientation in Melanoplus differentialis. Ten cells (five of each species) were studied, firstly in living cultures where chromosome behavior was followed, then by serial-section electron microscopy where the structural basis for chromosome behavior was examined. Microtubules other than kinetochore microtubules were observed impinging on the X chromosomes. One end of these microtubules was buried in chromatin, while the other ran towards a pole. The X chromosomes of M. sanguinipes had more of these microtubules than did M. differentialis X chromosomes. It is suggested that M. sanguinipes X chromosomes are less condensed than M. differentialis X chromosomes and so allow more microtubules to penetrate the chromosome. The extra microtubules impinging on the M. sanguinipes X chromosome probably prevent reorientation by inhibiting the turning of the chromosome towards the opposite pole, i.e., more force is needed to turn a kinetochore towards the opposite pole than can be generated and attempts at reorientation fail. This may be analogous to the effect that tension has on the orientation stability of bivalents.     

Malorientation in half-bivalents at anaphase in crane fly spermatocytes following Colcemid treatment

Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.     

Spindle microtubules and their mechanical associations after micromanipulation in anaphase

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

Kinetochore fibre dynamics outside the context of the spindle during anaphase

Chromosomes move polewards as kinetochore fibres shorten during anaphase. Fibre dynamics and force production have been studied extensively, but little is known about these processes in the absence of the spindle matrix. Here we show that laser-microbeam-severed kinetochore fibres in the cytoplasm of grasshopper spermatocytes maintain a constant length while turning over in a polarized manner. Tubulin incorporates at or near the kinetochore and translocates towards severed ends without shortening the fibre. Consequently, the chromosome cannot move polewards unless the severed fibre reattaches to the pole through microtubules. A potential seclusion artefact has been ruled out, as fibres severed inside spindles behave identically despite being surrounded by the spindle matrix. Our data suggest that kinetochore microtubules constantly treadmill during anaphase in insect cells. Treadmilling is an intrinsic property of microtubules in the kinetochore fibre, independent of the context and attachment of the spindle. The machinery that depolymerizes minus ends of kinetochore microtubules is functional in a non-spindle context. Attachment to the pole, however, is required to cause net kinetochore fibre shortening to generate polewards forces during anaphase.     

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.M.F. Trendelenburg     

Unipolar orientation stability of the sex univalent in the grasshopper (Melanoplus sanguinipes)

The behavior of the X chromosome in living Melanoplus sanguinipes primary spermatocytes was examined utilizing phase contrast microscopy and micromanipulation. During early prometaphase I, the X univalent establishes a unipolar orientation which is normally stable. In 28 cells, the X chromosome did not reorient once during a total of 122 h of observation. Though normally stable, the X chromosome can be induced to reorient by micromanipulation. An anomalous increase in X reorientation was observed in one testicular preparation. Stable unipolar orientation is an intrinsic characteristic of the X chromosome and was not displayed by two small autosomal univalents discovered in one cell. The two autosomal univalents, resulting from asynapsis or desynapsis, oscillated back and forth within the spindle, averaging 26.0 and 17.6 min respectively between reorientation events. The behavior of the M. sanguinipes X chromosome reveals an orientation stability mechanism that does not involve tension created by forces from opposing kinetochoric spindle fibers.     

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Timing of centrosome separation is important for accurate chromosome segregation

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not "search" the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.     

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

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

Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres

The motion of a chromosome during mitosis is mediated by a bundle of microtubules, termed a kinetochore fibre (K-fibre), which connects the kinetochore of the chromosome to a spindle pole. Once formed, mature K-fibres maintain a steady state length because the continuous addition of microtubule subunits onto microtubule plus ends at the kinetochore is balanced by their removal at their minus ends within the pole. This condition is known as 'microtubule poleward flux'. Chromosome motion and changes in position are then driven by changes in K-fibre length, which in turn are controlled by changes in the rates at which microtubule subunits are added at the kinetochore and/or removed from the pole. A key to understanding the role of flux in mitosis is to identify the molecular factors that drive it. Here we use Drosophila melanogaster S2 cells expressing alpha-tubulin tagged with green fluorescent protein, RNA interference, laser microsurgery and photobleaching to show that the kinetochore protein MAST/Orbit - the single CLASP orthologue in Drosophila - is an essential component for microtubule subunit incorporation into fluxing K-fibres.     

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells

The role of the kinetochore in chromosome movement was studied by 532- nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.     

Kinetochore capture and bi-orientation on the mitotic spindle

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.     

Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males

Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections. The majority (54%) of the microtubules extended from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole. Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%). We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore. Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position. For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90 degrees to the spindle axis may have microtubules running towards one pole only.     

Geometry and force behind kinetochore orientation: lessons from meiosis

During mitosis, replicated chromosomes (sister chromatids) become attached at the kinetochore by spindle microtubules emanating from opposite poles and segregate equationally. In the first division of meiosis, however, sister chromatids become attached from the same pole and co-segregate, whereas homologous chromosomes connected by chiasmata segregate to opposite poles. Disorder in this specialized chromosome attachment in meiosis is the leading cause of miscarriage in humans. Recent studies have elucidated the molecular mechanisms determining chromosome orientation, and consequently segregation, in meiosis. Comparative studies of meiosis and mitosis have led to the general principle that kinetochore geometry and tension exerted by microtubules synergistically generate chromosome orientation.