Dynamic changes in autosomal spindle fibers during prometaphase in crane fly spermatocytes

Different prometaphase stages of Pales ferruginea spermatocytes were serially sectioned and the regions between kinetochores and poles analysed by counting and measuring spindle microtubules. These regions are characterized by an intermingling of kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs). A considerable proportion of nkMTs is skewed with respect to kMTs, thus being responsible for microtubule disorder in these spindle areas. The degree of disorder expressed by the percentage of skew microtubules was found to decrease from early prometaphase to metaphase, parallel with an increase in kMT number. A possible causal relation between pulling forces and morphological changes in the spindle is discussed.     

Three-dimensional architecture of chromosome fibres in the crane fly: co-oriented autosomal bivalents and amphitelic sex univalents during prometaphase

A technique for the fixation of cells during live observation (Nicklas et al. 1979) was used to investigate chromosomes which were moving at the time of fixation. Chromosome fibres were reconstructed by tracking their microtubules in longitudinal serial sections. A considerable proportion of non-kinetochoric microtubules (free microtubules, fMTs) is skewed with respect to the fibre axis. These skew fMTs contribute to the degree of disorder. It was found that the difference in the relative proportion of skew fMTs between active fibres (oriented in the direction of movement) and passive fibres (oriented backwards) is significantly correlated with the chromosome velocity (correlation coefficient r=0.796, P=0.01). It can be concluded that the pulling force generated in the chromosome fibre is a function of skew fMTs.     

Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope

Prometaphase I chromosome behavior was examined in wild-type Drosophila melanogaster primary spermatocytes. Cine analysis of live cells reveals that bivalents exhibit complex motions that include (1) transient bipolar orientations, (2) simultaneous reorientation of homologous kinetochores, (3) movements not parallel to the spindle axis, and (4) movement along the nuclear membrane. — Kinetochores and kinetochore microtubule have been analyzed for bivalents previously studied in life. The results suggest that most chromosome motions (complex though they may be) can be explained by poleward forces acting on or through kinetochore microtubules that span the distance between the kinetochore and the vicinity of a pole. The results also suggest that the majority of short kinetochore microtubules may be remnants of previous microtubule-mediated associations between a kinetochore and a pole.     

Microtubule disorientation in anaphase half-spindles during autosome segregation in crane fly spermatocytes

The region between the kinetochores of syntelically oriented autosomes and the pole in meta- and anaphase of Pales ferruginea spermatocytes was studied by means of serial sections. Microtubule (MT) were counted and measured, and the spindle region was reconstructed by superimposition of successive micrographs. Kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs) interdigitate with one another forming a bundle which is often arrow-shaped due to an inclination of nkMTs (skew nkMTs) with respect to the kinetochore-pole axis. The average length of MT in the bundle decreases towards anaphase while the average number increases. The extent of MT disorder in anaphase half-spindles is higher than in metaphase. The number of kMTs inserted in the kinetochore was found to remain unchanged from meta- to early anaphase. Some of the kMTs become divergent in anaphase. The relative proportion of skew nkMTs within the kMT/nkMT bundle is higher in anaphase. It is proposed that the morphological changes observed to occur from meta- to anaphase are due to fragmentation of kMTs followed by disorientation of the MTs pieces. Some aspects of the physical properties of the half-spindles are discussed.     

The arrangement of microtubules and the attachment of chromosomes to the spindle during anaphase in tipulid spermatocytes

Analysis of serial sections oriented parallel to the interpolar spindle axis revealed the following results. Autosomes in anaphase of the 1. meiotic division of Pales ferruginea spermatocytes are attached to the spindle in two ways: 1. The short kinetochoric microtubules (kMTs) diverge and interdigitate with the axial mass of non-kinetochoric microtubules (nkMTs). 2. The chromosome surface shows projections which protrude between the mass of nkMTs. — At the level of anaphase plates the concentration of nkMTs is higher than in the interzone. — The lagging sex chromosomes at the equator become stretched by anaphase forces during autosomal movement. — The mean length of nkMTs in metaphase is 3.0±0.1 μm, in anaphase 2.6±0.1 μm, possibly indicating an overall MT shortening in anaphase. Spindle architecture and aspects of anaphase forces are discussed.     

Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold- recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold- recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.     

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.     

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.     

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

Microtubules of two chromosome fibres in metaphase and two in anaphase of the first meiotic division of Pales ferruginea spermatocytes were traced in serial sections. The number and lengths of microtubules constituting the fibres were determined and the fibres reconstructed. The chromosome fibres were found to contain only about 20% (in metaphase) and 9% (in anaphase) kinetochore microtubules (kMT), taking the sum of all individual microtubule lengths as the measure. The remainder consisted of free microtubules (fMT), a certain percentage of which were oriented obliquely with respect to the kMTs (skew fMTs). Between metaphase and anaphase most kMTs and fMTs shortened, causing a decrease in mean length of about onehalf in the case of kMTs. The proportion of skew fMTs increased considerably in anaphase, leading to more disorder in the fibre. The absolute mass of microtubules in the fibre seems to be irrelevant for spindle function. This can be deduced from the observed variability of absolute mass. A statistical correlation was found between the total mass of microtubules (kMTs+fMTs) and the number of kMTs, suggesting that some functional interrelation exists between these parameters.Dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

Morphological studies on the structure of univalent sex chromosomes during anaphase movement in spermatocytes of the crane fly Pales ferruginea

The ultrastructure of moving amphitelic sex chromosomes during anaphase of the first meiotic division of spermatocytes was studied by means of serial sections. The chromosomes have radial lamellae-like projections at their surface running in the direction of the spindle axis. Parallel spindle microtubules lie between these lamellae. The kinetochoral region pointing to the interzone (K_IZ) is stretched, while the kinetochore pointing polewards (K_p) is flat. It is suggested that the importance of the poleward kinetochore as an application site for pulling forces during anaphase movement is possibly reduced in favour of the lamellaemicrotubule association at the periphery of the chromosome. Distal parts of kinetochoral tubules of K_IZ may be associated with the lamellae of the partner sex chromosome. This arrangement may have some additional importance for the synchronization of sex chromosome migration in anaphase.     

Influence of autosome movements and of sex-chromosome movements on sex-chromosome segregation in crane fly spermatocytes

In crane fly spermatocyte meiosis 3 autosome half-bivalents normally move to each spindle pole in anaphase while the 2 amphitelic sex-chromosome univalents remain at the equator. The sex-chromosome univalents move to opposite poles after the autosomes reach the poles. — We used micromanipulation to detach half-bivalents in anaphase. When re-attached half-bivalents were syntelically oriented to the original pole, sex-chromosome segregation was usually not altered. When re-attached half-bivalents were amphitelically oriented, sex-chromosome segregation was usually altered: usually the amphitelic autosome segregated against one sex-chromosome while the other sex-chromosome remained at the equator. When re-attached half-bivalents were syntelically oriented to the opposite pole, sex-chromosome segregation was often altered: often one sex-chromosome moved normally to the spindle pole with 2 autosomal half-bivalents, while the other sex-chromosome did not move to the spindle pole with 4 autosomal half-bivalents, but remained at the equator. — The direction of motion of a sex-chromosome could be altered even after sex-chromosome segregation had begun, by suitable micromanipulation of the other sex-chromosome. — Amphitelic chromosomes that were not on the equator at the start of anaphase segregated predominantly to the closer spindle pole. Detached half-bivalents showed no preference for the closer pole when they re-attached with syntelic orientation. — We discuss some possible hypotheses for non-independent movements, and some implications of the results.     

Prophase chromosome movements in living house cricket spermatocytes and their relationship to prometaphase,anaphase and granule movements

Chromosome and granule movements in meiotic prophase and prometaphase have been studied by time-lapse cinemicrography in live spermatocytes of the house cricket, Acheta domesticus. Chromosome movements in prophase cells, up to one hour or more before breakdown of the nuclear envelope, are described. These movements are frequent but saltatory; are based mostly at chromosome ends but also at kinetochores; occur in very intimate association with the inside of the nuclear envelope; are directed towards and away from the extranuclear centres (centrioles); tend weakly to accumulate bivalents round the two centres and reach a velocity of 0.65 m/sec. Saltatory movements in granules associated with extranuclear asters are remarkably similar in basic characteristics to the intranuclear chromosome movements. Surprisingly, the chromosome movements (and those of granules) are reversably blocked by colcemid (but not lumi-colcemid), and yet occur in the apparent absence of an intranuclear microtubule array. The movements cease at or shortly after breakdown of the nuclear envelope. However, kinetochore movements in very early prometaphase are similar in velocity and other respects to prophase movements; later prometaphase movements are clearly slower, and those of anaphase very much slower still. — The prophase movements suggest a two component model for motion: a non-microtubule, linear force producer together with microtubules with a skeletal, orientational role. Arguably, both these components are also necessary for chromosome movements in prometaphase and anaphase.This paper is dedicated to Dr. Sally Hughes-Schrader, whose beautiful work in mantids clearly presaged the existence of chromosome movements in late prophase of meiosis; and whose enthusiasm over chromosome movements in general it was my pleasure to share during my stay at Duke.     

An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew)

Decorated actin-like filaments were seen in spindles after crane fly spermatocytes were glycerinated and then treated with rabbit skeletal muscle heavy meromyosin (HMM). Both ATP and pyrophosphate inhibited the HMM reaction. In prometaphase, metaphase, and mid-anaphase cells, actin-like filaments were seen near regions where chromosomal spindle fibres are seen in living cells, and were oriented in the pole-to-pole direction. In the interzone of anaphase cells, actin-like filaments were not oriented in a preferential direction when they were not associated with the microtubules attached to the sex chromosomes. No filaments were seen in glycerinated spindles not treated with HMM. We discuss reasons why filaments might not be seen without prior HMM treatment, and we discuss the possible role of the actin-like filaments in the spindles. — Spindle microtubules often were not seen in cells treated with HMM. This depended on the stage of division: in prometaphase no microtubules were seen; in metaphase microtubules were seen, in apparently normal numbers; in mid-anaphase, microtubules between the autosomes and the poles were seen in reduced numbers, those associated with the equatorial sex-chromosomes were seen in apparently normal numbers, while those between the separating autosomal half-bivalents were not seen. Microtubules were not seen in glycerinated spindles not treated with HMM, suggesting that HMM in some way affects microtubule stability. The question of microtubule stability is briefly discussed.     

An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew)

Actin-like filaments are seen at the cell periphery after crane fly spermatocytes are glycerinated and then treated with rabbit skeletal muscle heavy meromyosin. ATP and pyrophosphate inhibit the reaction with heavy meromyosin. From prometaphase through metaphase the filaments are all parallel to the cell surface, extending 0.5–1 μ. beneath the plasma membrane in a continuous layer of parallel filaments enveloping the cell; considering the poles of the spindle as north and south poles of the cell, the actin-like filaments at the cell periphery are all arranged as meridians. In late-anaphase, too, actin-like filaments are parallel to the cell surface, but here this includes bundles of filaments oriented as parallels in the furrow and adjacent regions of the cell periphery, as well as filaments oriented as meridians in the rest of the cell periphery. — Actin-like filaments are seen in the cellular projections associated with the spindle poles.     

Differential anaphase-I behaviour between wheat and rye univalents in triticale-wheat hybrid plants

The chromosomes of the D genome of wheat and the genome R of rye can be distinguished at meiosis by C-banding in triticale-wheat hybrid plants. All members of both genomes almost exclusively formed univalents at metaphase I. However, at anaphase I the frequencies of equationally dividing chromosomes were higher for rye than for wheat chromosomes. The differential centromere behaviour at anaphase I is ascribed to differences in the time at which wheat and rye univalents are formed during the first meiotic prophase.     

Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase

In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concurrently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region. To test the dynamic equilibrium model of chromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber microtubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated. Abrupt changes in temperature from 22 degrees C to as low as 4 degrees C or as high as 31 degrees C in fact produced immediate changes in chromosome velocity to new constant velocities. No transient changes in velocity were observed. At 4 degrees C (10 degrees C for grasshopper cells), chromosome movement ceased. Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little. The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between temperature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber microtubules. On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways of polymerization and depolymerization.     

Arrangement and dynamics of spindle structures in crane fly spermatocytes seen with video-enhanced contrast differential interference contrast microscopy

This is a report on the arrangement and dynamic behaviour of microtubule fibres investigated by video-intensified microscopy (Allen Video Enhanced Contrast —Differential Interference Contrast, AVEC-DIC) in spindles of living crane fly spermatocytes. The spindle was found to contain numerous fibrils, each fibril probably consisting of several microtubules. The fibrils are oriented between the poles and, due to slight inclinations, towards each other, frequently form arrowhead-like structures or points of intersection. This leads to an overall lattice-like arrangement. The fibrils display flickering motions visible in real time recordings. The observations are discussed in relation to ultrastructural data on chromosome fibre architecture from previous studies.     

The behaviour of chromosomal axes during diplotene in mouse spermatocytes

The fate of the synaptonemal complex and its elements after pachytene has been studied by serial sectioning of diplotene nuclei in mouse spermatocytes. The lateral elements of the synaptonemal complex separate from each other during diplotene, and they form single axes, 300 Å wide, surrounded by chromatin fibrils. The single axes are continuous and end on the nuclear membrane by two different ends: the basal knob and the simple end. The single axes do not cross-over each other, but they remain approached at the convergence regions. In these regions a modified piece of synaptonemal complex is found. This piece changes into a chromatin bridge during diplotene. It has been inferred that the convergence regions represent chiasmata and that the single axes do not represent axial structures of chromatids.