Sex-chromosome anaphase movements in crane-fly spermatocytes are coordinated: Ultraviolet microbeam irradiation of one kinetochore of one sex chromosome blocks the movements of both sex chromosomes

Various aspects of chromosome movements in cranefly spermatocytes are coordinated (Forer, 1982a), by which we mean that the movements of spatially separate chromosomes are ‘linked’ in an interdependent manner. Coordination between the sex chromosomes is seen in several circumstances, as follows. In normal metaphase and anaphase crane-fly spermatocytes each of the two sex-chromosome univalents is oriented amphitelically (i.e. each has chromosome spindle fibres to both poles) and each moves polewards only after the autosomes have reached the poles (Bauer et al. 1961). The two sex chromosomes begin to move polewards at the same time, even though they are not paired and are physically separated; thus sex-chromosome movements are coordinated in timing. Sex-chromosome movements also are coordinated in direction, since the sex chromosomes very rarely, if ever, move to the same pole; this directional coordination continues even after sex chromosomes have began to move to opposite poles, as demonstrated by the following experiment. When a sex chromosome moving to one pole is pushed in the opposite direction, past the chromosome moving to the opposite pole, both sex chromosomes reverse direction of motion and move to new poles (Forer & Koch, 1973). Thus there are coordinations in timing and direction of sex-chromosome movements, and the coordinations in direction continue throughout anaphase.

There also seem to be mechanisms that take into account (that count) the numbers of amphitelically oriented chromosomes. When there is only one amphi-telic chromosome, that chromosome does not move from the equator; when there are two, one moves to each pole; when there are three, two move (to opposite poles) and one remains at the equator; when there are four, two move to each pole (Dietz, 1969; Forer & Koch, 1973). Thus the amphitelically oriented chromosomes seem to be ‘counted’.

One might argue that these examples do not really indicate that there are systems for ‘coordination’, but rather that the presumed ‘coordinations’ are built into the force production system, i.e. that the force production system requires one sex chromosome to move against another. The following results rule out this argument.

In some circumstances the sex chromosomes can move singly. When there are four autosomes at one pole and two at the other (achieved either in non-treated cells (Dietz, 1969) or via micromanipulation (Forer & Koch, 1973)) one sex chromosome moves to the pole that has two autosomes and the other remains at the equator. In other experiments, when an autosomal spindle fibre that is adjacent to one sex-chromosome’s spindle fibre is irradiated with an ultraviolet microbeam, one sex chromosome subsequently moves polewards (towards the pole associated with the nonirradiated half-spindle) while the other remains at the equator (Sillers & Forer, 1981). Thus one sex chromosome can move alone.

In some circumstances both sex chromosomes can move to the same pole. This occurs after microbeam irradiation of an autosomal spindle fibre that is adjacent to two sex-chromosomal spindle fibres: both sex chromosomes move to the same pole, which is that associated with the half-spindle that was not irradiated (Sillers & Forer, 1981). Thus, the force production system does not require that sex chromosomes move to opposite poles: the sex chromosomes can move singly or both can move to the same pole.

In summary, there seem to be ‘coordination’ mechanisms or ‘signalling’ systems that control directions and timing of sex-chromosome motions in normal crane-fly spermatocytes; there seem to be mechanisms for counting the number of sex chromosomes (to determine how many will move); and there seem to be mechanisms for determining whether or not sex chromosomes will move, and in which directions, depending on the previous segregation of autosomes and on the state of the adjacent autosomal spindle fibres.

We report here experiments in which we studied the coordination between sex chromosomes in crane-fly spermatocytes by irradiating sex-chromosomal kineto-chores with an ultraviolet (u.v.) microbeam. We wanted to see if inactivation of one of the two kineto-chores of a sex chromosome would influence the movement of the irradiated chromosome and to see if alterations in the movement of the irradiated chromosome would influence the other sex chromosome. Kinetochores have been inactivated previously using microbeam irradiations. For example, in newt cells ‘centrophilic’ chromosomes did not congress to the metaphase plate after their kinetochores were irradiated with a u.v. microbeam (Uretz et al. 1954; Bloom et al. 1955). In grasshopper spermatocytes (Izutsu, 1959, 1961), Haemanthus endosperm (Bajer & Mole-Bajer, 1961; Bajer, 1972), and newt cells (Uretz et al. 1954; Bloom et al. 1955) anaphase motions were blocked after kinetochores were irradiated with a u.v. microbeam. In tissue-culture cells, chromosome movements were blocked after a laser microbeam was used to ablate kinetochores (McNeil & Berns, 1981; Reider et al. 1986). Thus microbeam irradiations of kinetochores can inactivate kinetochores and block subsequent movements of the irradiated chromosomes.

In this paper we describe the results of ultraviolet microbeam irradiations of sex-chromosome kinetochores in spermatocytes of the crane fly Nephrotoma abbreviata (Loew). We used N. abbreviata spermatocytes, because in this species the sex chromosomes are so large that we are able to irradiate one of the two kinetochores without hitting the second. Irradiations in anaphase of one kinetochore, of either the X-chromo-some or the Y-chromosome, permanently stopped motion of both chromosomes. We argue that this is due to a coordination system that involves interactions between chromosomal spindle fibres.

Crane-fly spermatocytes were prepared from 4th instar larvae of a laboratory colony (Nephrotoma abbreviata (Loew)) using described preparation and rearing methods (Forer, 1982b). For preparations in which we did not irradiate cells, a flamed glass coverslip was taped over a 23 mm diameter hole in an aluminium slide. The slide plus coverslip formed a well that was filled with Halocarbon oil (series 700; Halocarbon Products, Hackensack, NJ). Testes were immersed in the oil and individual cells spread on the coverslip. Spermatocvte preparations were observed using phase-contrast microscopv (100×, NA= 1·25, Carl Zeiss). For preparations in which we irradiated cells, we used quartz coverslips, 0·35 mm thick (Applied Physics Specialties, Toronto); quartz coverslips were re-used after washing with soap and rinsing with distilled water.

Spermatocytes were irradiated using a Reichert Biovert inverted microscope adapted for u.v. microbeam irradiations, as described (Wilson & Forer, 1987). Briefly, the u.v. light used for irradiations passes through a quartz lens and a shutter, and into a monochromator. The exit slit from the monochromator is focused onto a pinhole, which is then focused by a Carl Zeiss 100× Ultrafluar phase-contrast objective (glycerine immersion, NA = 0·85) through a quartz coverslip onto the specimen. In our experiments, all irradiations were with light of wavelength 280 nm, and were for 90s in a spot 1·2 μm in diameter (in the cell), giving total irradiation doses of about 0’47 ergs μm⁻².

Living spermatocytes were observed using a Panasonic model WV-1500 TV camera and videotaped with a Panasonic model 6300 video casette recorder. Photographs were taken from the video monitor using a Nikon F3 camera, with the tape running. Measurements were made from the video screen; quantitative data are presented as averages ± standard deviations. In one case (Fig. 1), cells were photographed directly through the microscope, using standard photographic techniques.

We describe normal sex-chromosome movements during meiosis in N. abbreviata spermatocytes prior to describing the effects of irradiation.

At anaphase, autosomal half-bivalents separate and move to opposite poles, during which time the two sexchromosome univalents remain at the equator; the sex chromosomes move poleward only after the autosomes have reached the poles, as in spermatocytes of other species (Henderson & Parsons, 1963; Dietz, 1969; Forer, 1982b). In Nephrotoma abbreviata, when a sex chromosome is in one plane of focus it is shaped roughly as a ‘cylinder’, 8 ± 2·3 μm long × 3·5 ± 1·2 ·m wide (n = 20). The sex-chromosome cylinders often appear bent from one point, i.e. each has two arms, joined at the kinetochore. When bent, one sex chromosome is J-shaped and the other is U-shaped, so one sex chromosome is metacentric and the other is acrocentric. Even when the two arms are not askew the kinetochore regions can be recognized at anaphase as stretched-out regions of the chromosome (Fig. 1).

The two sex chromosomes had various orientations during anaphase. In the usual case each sex chromosome moved with its long axis perpendicular to the spindle axis (Figs 5, 7). In some cells, however, one of the sex chromosomes moved with its long axis parallel to the spindle axis (see Fig. 9). We assume that when the sex-chromosome axis is perpendicular to the spindle axis the sex chromosome is amphitelically oriented, as are sex chromosomes in spermatocytes of other species of crane flies (Bauer et al. 1961; Fuge, 1973; Steffen & Fuge, 1985; Janicke & LaFountain, 1984), i.e. each kinetochore in the sex chromosome is connected to a different pole, the two kinetochores being connected to opposite poles. We assume that when the sex-chromosome axis is parallel to the spindle axis the sex chromosome is merotelically oriented, i.e. each kinetochore in the sex chromosome is connected to both poles. (As described by Janicke & LaFountain (1984), individual kinetochores of half-bivalents in crane-fly spermatocytes can be induced to become merotelically oriented, with microtubules from individual kinetochores extending to both poles.) In our descriptions, a sex chromosome that moved with its long axis perpendicular to the spindle axis will be referred to as being normally oriented (amphitelic) and one that moved with its long axis parallel to the spindle axis will be referred to as being maloriented (mcro-telic).

We quantified various aspects of anaphase. For separating half-bivalents we measured the inter-kineto-chore distances and constructed graphs of distance versus time. For separating sex chromosomes we measured the distances (in the direction of the spindle axis) between the two closest kinetochores and plotted distance versus time (on the same graphs as the autosomes). The curves for separating autosomal halfbivalents are usually S-shaped (Figs 2, 3). Prior to anaphase the inter-kinetochore distance remains constant; the half-bivalents separate at anaphase and after a short period of acceleration the autosomes move poleward for 10–15 min with constant velocity of separation of about 1 μm min⁻¹, and with no spindle elongation. Once the autosomes have reached the poles the curves level off and further increase in distance between autosomal half-bivalents is due to spindle elongation. Thus, the spindle stays at a constant length when the autosomes move polewards, but spindle length increases after the autosomes near the poles.

The movements of sex-chromosome univalents are different from those of the autosomes. When both sex chromosomes are normally oriented the sex-chromosome movements generally have three phases: there is an initial slower velocity (of 0·3 ± 0·1 μm min⁻¹, n = 9) for the first 5 min, a later, faster velocity (of 0·6 ± 0·2 μm min⁻¹ = 9) for the next 15 min, and then the velocities fall off, becoming near zero when the sex chromosomes reach the poles. Increases in inter-kinetochore distances after this time are due to spindle elongation (Fig. 2). When one sex chromosome is maloriented, on the other hand, sex-chromosome movements do not have the three phases; rather, after what appears to be an initial, faster separation lasting a few minutes, the sex chromosomes separate at a constant, slower velocity (Fig. 3) of 0·4 ± 0·3 μm min⁻¹ (n = 18).

For the various aspects of anaphase that we quantified, we compared spermatocytes that had two normally oriented sex chromosomes with those in which one sex chromosome was maloriented. (We did not observe any cells in which both sex chromosomes were maloriented.) As summarized in Table 1, sex-chromosome orientation does not appear to affect the parameters that were measured, except for the final distance of the sex chromosomes from the poles, measured from the poleward side of the autosomes when cleavage began: normally oriented sex chromosomes generally reach the poles before the cells begin to cleave, whereas maloriented sex chromosomes rarely do so. (Velocities for autosome and sex-chromosome movements were obtained from the steepest part of each graph of distance versus time, using the least-mean-squares method.)

The initial microbeam experiments were done to see if motion would stop after we irradiated one sex-chromosome kinetochore in anaphase. The kinetochore regions, the pulled out areas of the chromosomes, or, when the chromosomes were bent, the ‘indentations’ at the junctions of the chromosome arms, were identified ; then, in sex-chromosome anaphase, we irradiated one of the two kinetochores of the univalent in question.

We first irradiated cells in which both sex chromosomes were oriented normally. Irradiations usually resulted in ‘paling’ of the chromosome in the area in which the ultraviolet light was focused (Fig. 4), as described originally in newt cell chromosomes (Uretz et al. 1954; Bloom et al. 1955). The paling occurred during the 90s of the irradiation and later spread to include 52(± 18)% (n = 12) of the total length of the chromosome. After spreading, the paling was not localized to the irradiated chromatid, and often included the non-irradiated kinetochore. Following paling, the entire chromosome returned to its original optical density (i.e. the chromosome recovered), usually within minutes after the irradiation. The paling verified which region of the chromosome we had irradiated.

After irradiation of one sex chromosome’s leading kinetochore, i.e. the kinetochore closest to the pole towards which the chromosome was moving, the movements of both sex chromosomes were blocked; movements of both chromosomes ceased and never recovered. There was sometimes ‘apparent’ movement of sex chromosomes at 10 min or more after the irradiations ceased, as indicated by a non-zero slope on the graphs of distance versus time, but these movements occurred only at the time of cell cleavage, and we assume that they were caused by the cleavage furrows. There were only three exceptions: in three cells motion of both sex chromosomes was temporarily blocked, for 7 ± 1·2min, after which the univalents resumed movement at separation velocities of 0·4 ± 0·1 μm min⁻¹. We assume that there was incomplete inactivation of the kinetochores in these three cells and that complete inactivation results in permanent cessation of movement.

We tested whether other irradiations also blocked sex-chromosome motion. Irradiation of the trailing kinetochores blocked movement of both sex chromosomes (Figs 5, 6). Irradiation of spindle fibres at 2gm from the kinetochores either had no effect or temporarily blocked movement of both sex chromosomes (Figs 7, 8), for 8 min or less. Irradiation of the non-kinetochore part of the sex-chromosome did not block movement of either sex chromosome. Therefore, only irradiations of kinetochores permanently block movement, and when movements were blocked both sexchromosomes stopped moving.

We then irradiated cells with maloriented sexchromosomes. When we irradiated one kinetochore of the maloriented chromosome, chromosome movement was not blocked (Figs 9, 10). When wc irradiated one kinetochore of the normal sex chromosome, motion of both chromosomes was blocked, as in cells in which both sex chromosomes were oriented normally.

Velocity and distance parameters for all irradiations are summarized in Table 2, and the lengths of time for which chromosome movement was stopped are summarized in Fig. 11.

Irradiation of one sex-chromosomal kinetochore permanently stopped movement of both sex chromosomes in crane-fly spermatocytes, irrespective of which kinetochore was irradiated. Control irradiations of either the spindle fibre of one sex chromosome or the non-kinetochore part of the chromosome did not affect motion, or blocked motion only temporarily. Thus, in order to stop movement of the two sex chromosomes permanently, the irradiation must include the kinetochore region. Since one sex chromosome can move to a pole while the other sex chromosome remains at the equator (Dietz, 1969; Forer & Koch, 1973; Sillers & Forer, 1981), as summarized in the Introduction, blocking the movement of both sex chromosomes is not due to effects on the force-production system. Rather, these results would seem to be due to effects on a communication (or coordination or signalling) system: irradiation of one kinetochore of one sex chromosome causes a signal to be sent to the force-production system associated with the other sex chromosome, and that signal causes cessation of force production for the second sex chromosome. (Similar signals were identified in Neocurtilla (Grillotalpa) spermatocytes: irradiation of one chromosomal spindle fibre associated with the heteromorphic bivalent caused force production to cease both from that spindle fibre and from the spindle fibre directed to the opposite pole (Sillers et al. 1983).) What might the putative signal be?

We do not know the primary lesion produced by the ultraviolet light. If, however, we assume that the irradiation of a kinetochore inactivates that kinetochore and causes loss of the associated spindle fibre, as in other cells (described in the Introduction), then the coordination system between sex chromosomes in crane-fly spermatocytes seems to require that both sex chromosomes maintain spindle fibres to both poles in order for proper movements to occur. This interpretation is based on the following argument, which is illustrated in Fig. 12. Consider a cell in which both sex chromosomes are oriented normally (Fig. 12A). After irradiation of a kinetochore one of the four chromosomal spindle fibres is missing, and both sex chromosomes stop moving (Fig. 12C). The situation is different when one chromosome is maloriented, however; in that case, the maloriented chromosome has two sets of spindle fibres to each pole: one set to each pole from each kinetochore (Fig. 12B). Irradiation of one of the kinetochores of a maloriented chromosome would eliminate one set of fibres (Fig. 12D) but connections to both poles would still be maintained by the other kinetochore. Hence that chromosome would still have connections to both poles after the irradiations and such irradiations do not block sex-chromosome movements. This is not simply a matter of having at least two chromosomal spindle fibres to each pole, because irradiation of one kinetochore of the normally oriented sex chromosome blocks movement of both sex chromosomes, even though the maloriented chromosome maintains two sets of fibres to each pole (Fig. 12E). Thus a normal signal that allows both sex chromosomes to move seems to require that each chromosome must have a spindle fibre to each pole.

We do not know what the fibre-related signals might be, but two possibilities have been raised in the literature. One is that the signals (the coordinations) involve interactions of some kind between adjacent chromosomal spindle fibres (see discussion by Sillers & Forer, 1981). The other possibility is that the centrioles at the spindle poles act as receivers of signals and as interpreters, and relay instructions via spindle fibre microtubules, an extension of the putative role of centrioles in cell motility (Albrecht-Beuhler, 1985). The role of the centriole can be tested by repeating our experiments in cells in which there are no centrioles at the poles (e.g. see Dietz, 1969; Steffen et al. 1986).

Irradiations of the kinetochore usually caused paling of the chromosome in the irradiated area. This paling, similar to the paling reported previously in newt cells in culture (Uretz et al. 1954; Bloom et al. 1955; Perry, 1956), allowed us to confirm that we indeed irradiated where we thought we did. We took all the precautions described by Sillers & Forer (1981), Sillers et al. (1983) and Wilson & Forer (1987) to ensure that the u.v. microbeam was focused where we aimed it, and the paling provided further assurance that our aiming was correct. What is the paling due to?

Various workers have argued that the primary lesion in paling is in DNA, or in DNA plus protein (Perry, 1956; Bloom & Leider, 1962; Zirkle & Uretz, 1963; Setlow & Setlow, 1962). u.v.-induced lesions in DNA induce decondensation along the entire chromosome when repair of the lesion is prevented, and the chromosomes condense again when repair is permitted (Mull-inger & Johnson, 1987). The paling we see could be similar: microbeam-induced lesions induce local decondensation, this spreads until the lesions are repaired, and then the paling disappears as the chromosome condenses again. Indeed, Moreno (1971) has shown that lesions in DNA induced by u.v. microbeam irradiations are repaired in living cells. Alternatively, the effect might be mediated through a topoisomerase. Topoisomerases cause relaxation of tightly coiled DNA, so one could imagine that the paling we see is due to u.v. inactivating (and removing) a DNA-binding protein in the irradiated region and that loss of this protein permits topoisomerases to decondense the chromosome. The different interpretations might be tested through the use in vivo of inhibitors of DNA repair, or of antibodies to topoisomerases such as described by Guldner et al. (1986).

The work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the J. P. Bickell Foundation and the Atkinson Foundation. The York University Department of Physical Plant pesticide spraying programme provided us with enforced periods of contemplation, free from pressures (or even ability) to take data. We are grateful to Drs Donna Kubai and Dwayne Wise for sending us eggs of N. abbreviata, to help us keep the stocks going when we had high mortality. We are also grateful to Paula Wilson for her help with the microbeam.