Ultraviolet microbeam irradiation of chromosomal spindle fibres in Haemanthus katherinae endosperm. I. Behaviour of the irradiated region

1993 ◽  
Vol 105 (2) ◽  
pp. 571-578 ◽  
Author(s):  
B.B. Czaban ◽  
A. Forer ◽  
A.S. Bajer
Keyword(s):  
Ultraviolet Light ◽  
Spindle Fibre ◽  
Microbeam Irradiation ◽  
Ultraviolet Microbeam ◽  
Spindle Fibres

We used an ultraviolet microbeam to irradiate chromosomal spindle fibres in metaphase Haemanthus endosperm cells. An area of reduced birefringence (ARB) was formed at the position of the focussed ultraviolet light with all wavelengths we used (260, 270, 280, and 290 nm). The chromosomal spindle fibre regions (kinetochore microtubules) poleward from the ARBs were unstable: they shortened (from the ARB to the pole) either too fast for us to measure or at rates of about 40 microns per minute. The chromosomal spindle fibre regions (kinetochore microtubules) kinetochore-ward from the ARBs were stable: they did not change length for about 80 seconds, and then they increased in length at rates of about 0.7 microns per minute. The lengthening chromosomal spindle fibres sometimes grew in a direction different from that of the original chromosomal spindle fibre. The chromosome associated with the irradiated spindle fibre sometimes moved off the equator a few micrometers, towards the non-irradiated half-spindle. We discuss our results in relation to other results in the literature and conclude that kinetochores and poles influence the behaviour of kinetochore microtubules.

Analysis of chromosome movement in crane fly spermatocytes by ultraviolet microbeam irradiation of individual chromosomal spindle fibres. I. General results

1981 ◽  
Vol 59 (9) ◽  
pp. 770-776 ◽  
Author(s):  
Peggy J. Sillers ◽  
Arthur Forer
Keyword(s):  
Ultraviolet Light ◽  
Monochromatic Light ◽  
The Other ◽  
Spindle Fibre ◽  
Chromosome Movement ◽  
Opposite Pole ◽  
Other Hand ◽  
Ultraviolet Microbeam ◽  
The Difference ◽  
Spindle Fibres

Single chromosomal spindle fibres in anaphase Nephrotoma ferruginea (crane fly) spermatocytes were irradiated with monochromatic ultraviolet light focussed to a 4-μm spot by means of an ultraviolet microbeam apparatus. The movement of the half-bivalent associated with the irradiated spindle fibre was either unaffected or the half-bivalent stopped moving; i.e., the effect was all-or-none. When the half-bivalent associated with the irradiated spindle fibre did stop moving, the partner half-bivalent moving towards the opposite pole (i.e., the half-bivalent with which the first half-bivalent was previously paired) also stopped moving: all other half-bivalents moved normally. In over 90% of the 69 cases the movements of the two half-bivalents were only temporarily blocked; when movement resumed both half-bivalents resumed movement at the same time, after stoppage times ranging from 2 min to more than 15 min. In a few cases the half-bivalents never resumed poleward motion.When half-bivalents that had stopped movement finally resumed movement they often did not reach the poles; i.e., they "lagged" and remained separate from the other chromosomes. This result occurred only in spermatocytes of N. ferruginea. In spermatocytes of N. suturalis or N. abbreviata, on the other hand, the stopped half-bivalents did not lag but always reached the poles.Half-bivalent pairs that stopped moving in N. ferruginea spermatocytes did so for shorter times than did those previously reported (after irradiation of chromosomal spindle fibres) in N. suturalis spermatocytes. We suggest that the difference is due to our use of monochromatic ultraviolet light as opposed to the previous use of heterochromatic ultraviolet light. We assume that different wavelengths of monochromatic light produce different effects, that any given monochromatic irradiation produces only one effect (albeit different effects at different wavelengths), but that heterochromatic irradiations can produce multiple effects.Irradiation of the interzone (between separating half-bivalents) had no effect on the chromosome-to-pole movements of the half-bivalents. Therefore the stoppage of movement of half-bivalent pairs is specific for irradiation of chromosomal spindle fibres. On the other hand, irradiation of the interzone often blocked pole-to-pole elongation.

Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo

1988 ◽  
Vol 91 (4) ◽  
pp. 455-468 ◽  
Author(s):  
P.J. Wilson ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Relative Stability ◽  
Dynamic Instability ◽  
Spindle Fibre ◽  
Microtubule Depolymerization ◽  
Immunofluorescence Analysis ◽  
Ultraviolet Microbeam ◽  
Spindle Fibres

Irradiation of birefringent chromosomal spindle fibres in crane-fly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dynamic instability. Possible models for ARB motion are discussed.

Non-random chromosome segregation in Neocurtilla hexadactyla is controlled by chromosomal spindle fibres: an ultraviolet microbeam analysis

1984 ◽  
Vol 69 (1) ◽  
pp. 1-17
Author(s):  
D. Wise ◽  
P.J. Sillers ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Chromosome Segregation ◽  
Sex Chromosome ◽  
Spindle Fibre ◽  
Single Sex ◽  
Microbeam Analysis ◽  
Ultraviolet Microbeam ◽  
Random Segregation ◽  
Spindle Fibres

Single spindle fibres of Neocurtilla spermatocytes were irradiated by means of an ultraviolet microbeam. Irradiations were with monochromatic ultraviolet light. The single sex chromosome (the X1 univalent) reoriented after irradiation of its spindle fibre or of any of the spindle fibres associated with the heteromorphic bivalent (the X2Y bivalent): the X1 moved toward the Y half-spindle, and sometimes rotated as it moved. Irradiations of autosomal spindle fibres did not induce X1 movements, and hence the induction of reorientation is specific to irradiation of the spindle fibres associated with X1 or X2Y. In no case did the X2Y bivalent reorient; hence, the X1 is the active chromosome in ensuring that there is non-random segregation in Neocurtilla spermatocytes. The irradiations sometimes caused the X2Y bivalent to contrast, but the reorientation movements of the X1 were independent of the contraction of the X2Y bivalent. We suggest that the X1 and X2Y chromosomal spindle fibres form a network that is able to send signals to the X1 univalent to cause it to reorient.

Action spectrum for changes in spindle fibre birefringence after ultraviolet microbeam irradiations of single chromosomal spindle fibres in crane-fly spermatocytes

1983 ◽  
Vol 62 (1) ◽  
pp. 1-25
Author(s):  
P.J. Sillers ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Action Spectrum ◽  
The Other ◽  
Spindle Fibre ◽  
Chromosome Movement ◽  
Ultraviolet Microbeam ◽  
Two Peaks ◽  
Wavelength Light ◽  
Spindle Fibres ◽  
In Cells

Single chromosomal spindle fibres in Nephrotoma suturalis (crane-fly) spermatocytes in metaphase and anaphase were irradiated with monochromatic ultraviolet light focussed to a 2 micrometer spot. In cells in both metaphase and anaphase either the birefringence of the irradiated spindle fibre was altered in the irradiated region, or there was no change, depending on the dose and wavelength of ultraviolet light used for the irradiation. When there was an area of reduced birefringence (ARB), it moved poleward regardless of whether the associated chromosome moved poleward. When cells were irradiated in early metaphase they remained in metaphase until the ARB reached the pole. In some cells irradiated in late metaphase the chromosomes began anaphase before the ARB reached the pole; in many such cells anaphase was abnormal in that all six half-bivalents separated at the start of anaphase but none moved polewards. In all cases the ARB moved poleward at the same speed as subsequent chromosome movement; that is, at about 0.8 micrometer/min. In cells irradiated in anaphase, spindle fibre birefringence was reduced independently of blockage of chromosome movement. Because birefringence and movement were altered independently there were four classes of results: (1) in some cases there was no effect on the movement of the chromosome associated with the irradiated spindle fibre and no effect on the birefringence of the irradiated spindle fibre. (2)In some cases, primarily with 260 nm wavelength light, there was no effect on the movement of the chromosome associated with the irradiated spindle fibre and there was an effect on the birefringence of the irradiated spindle fibre. (3) In some cases, primarily with 290 nm wavelength light, there was an effect on the movement of the chromosome associated with the irradiated spindle fibre and no effect on the birefringence of the irradiated spindle fibre. (4) In some cases, primarily with 270 nm and 280 nm wavelength light, there was an effect on the movement of the chromosomes associated with the irradiated spindle fibre and there was an effect on the birefringence of the irradiated spindle fibre. The action spectrum for reducing spindle fibre birefringence in crane-fly spermatocytes had two peaks, one at 260 nm and the other, less sensitive, at 280 nm. For irradiations at 270 nm, 280 nm and 290 nm, five to fifty times more energy was needed to reduce spindle fibre birefringence than to stop chromosome movement, but for irradiations at 260 nm five times less energy was needed to reduce spindle fibre birefringence than to stop chromosome movement. The action spectrum for reducing spindle fibre birefringence is quite different from that for stopping chromosome movement.

The behaviour of microtubules in chromosomal spindle fibres irradiated singly or doubly with ultraviolet light

1989 ◽  
Vol 94 (4) ◽  
pp. 625-634
Author(s):  
P. Wilson ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Single Fibre ◽  
The Other ◽  
Microbeam Irradiation ◽  
Other Hand ◽  
Ultraviolet Microbeam ◽  
Spindle Fibres

Areas of reduced birefringence (ARBs) produced by ultraviolet microbeam irradiation are areas of depolymerized microtubules. ARBs probably move poleward either by microtubule subunit addition at the kinetochore and loss at the pole, or by microtubule subunit addition at one edge of the ARB and loss from the other edge. In this paper we have used two approaches to try to distinguish between these two models. First, we determined whether the edges of the ARB move at the same rate; if ARB motion is due solely to addition at the kinetochore and loss at the pole, with the ARB edges unable to exchange subunits, then the two edges of each ARB should move at the same rate. On the other hand, if the exchange is at the ARB edges, then, from data from microtubules in vitro, the poleward edge should move much faster than the kinetochoreward edge. We found that the two edges of the ARB move at the same rate about half the time, but half the time they do not. Second, we studied the behaviour of two ARBs on a single fibre. If ARB motion is due solely to subunit addition at the kinetochore and loss at the pole, then the two ARBs must move poleward together. We found that after two ARBs are formed on a single fibre the region between the ARBs is unstable and rapidly depolymerizes. These results do not fit either model and suggest that influences of kinetochores and poles or other factors need to be considered that are not duplicated in experiments on microtubules in vitro.

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

1987 ◽  
Vol 88 (4) ◽  
pp. 441-452
Author(s):  
JULIA A. M. SWEDAK ◽  
ARTHUR FORER
Keyword(s):  
Sex Chromosomes ◽  
Sex Chromosome ◽  
The Other ◽  
Signal System ◽  
Spindle Fibre ◽  
Spindle Axis ◽  
Ultraviolet Microbeam ◽  
Block Motion ◽  
Block Movement ◽  
Spindle Fibres

Sex chromosomes in crane-fly spermatocytes move polewards at anaphase after the autosomes have reached the poles. In Nephrotoma abbreviate the sex chromosomes are 8 μm long by 3.5 μm wide and have two orientations when they move: the long axis of the sex chromosome is either perpendicular or parallel to the spindle axis. We assume (1) that when a sex chromosome is perpendicular to the spindle axis it has a chromosomal spindle fibre to each pole, one from each kinetochore, as in other species; and (2) that when a sex chromosome is parallel to the spindle axis each kinetochore has spindle fibres to both poles, i.e. that the latter sex chromosomes are maloriented. We irradiated one kinetochore of one sex chromosome using an ultraviolet microbeam. When both sex chromosomes were normally oriented, irradiation of a single kinetochore permanently blocked movement of both sex chromosomes. Irradiation of non-kinetochore chromosomal regions or of spindle fibres did not block movement, or blocked movement only temporarily. We argue that ultraviolet irradiation of one kinetochore blocks movement of both sex chromosomes because of effects on a ‘signal’ system. The results were different when one sex chromosome was maloriented. Irradiation of one kinetochore of a maloriented sex chromosome did not block motion of either sex chromosome. On the other hand, irradiation of one kinetochore of a normally oriented sex chromosome permanently blocked motion of both that sex chromosome and the maloriented sex chromosome. We argue that for the signal system to allow the sex chromosomes to move to the pole each sex chromosome must have one spindle fibre to each pole.

Does actin produce the force that moves a chromosome to the pole during anaphase?

1985 ◽  
Vol 63 (6) ◽  
pp. 585-598 ◽  
Author(s):  
Arthur Forer
Keyword(s):  
Spindle Fibre ◽  
Apparent Contradiction ◽  
Spindle Poles ◽  
Ultraviolet Microbeam ◽  
The Face ◽  
Spindle Fibres ◽  
Rule Out ◽  
Do So

Chromosomes move towards spindle poles because of force produced by chromosomal spindle fibres. I argue that actin is involved in producing this force. Actin is present in chromosomal spindle fibres, with consistent polarity. Physiological experiments using ultraviolet microbeam irradiations suggest that the force is due to an actin and myosin (or myosin-equivalent) system. Other physiological experiments (using inhibitors in "leaky" cells or antibodies injected into cells) that on the face of it would seem to rule out actin and myosin on closer scrutiny do not really do so at all. I argue that in vivo the "on" ends of chromosomal spindle fibre microtubules are at the kinetochores; I discuss the apparent contradiction between this conclusion and those from experiments on microtubules in vitro. From what we know of treadmilling in microtubules in vitro, the poleward movements of irradiation-induced areas of reduced birefringence (arb) can not be explained as treadmilling of microtubules: additional assumptions need to be made for arb movements toward the pole to be due to treadmilling. If arb movement does indeed represent treadmilling along chromosomal spindle fibre microtubules, treadmilling continues throughout anaphase. Thus I suggest that chromosomal spindle fibres shorten in anaphase not because polymerization is stopped at the kinetochore (the on end), as previously assumed, but rather because there is increased depolymerization at the pole (the "off" end).

scholarly journals Autosomal spindle fibres influence subsequent sex-chromosome movement in crane-fly spermatocytes

1981 ◽  
Vol 49 (1) ◽  
pp. 51-67 ◽  
Author(s):  
P.J. Sillers ◽  
A. Forer
Keyword(s):  
Control System ◽  
Sex Chromosomes ◽  
Sex Chromosome ◽  
Complete Recovery ◽  
Wavelength Dependence ◽  
Spindle Fibre ◽  
Ultraviolet Microbeam ◽  
Direct Irradiation ◽  
Chromosome Movements ◽  
Spindle Fibres

In meiosis-I crane-fly spermatocytes 3 autosomal half-bivalents move to each pole in anaphase while the 2 sex-chromosomal univalents remain at the equator. The sex chromosomes move to opposite poles only after the autosomes reach the poles; the sex chromosomes start to move polewards about 25 min after the autosomal half-bivalents have begun to move. We irradiated portions of single autosomal spindle fibres with an ultraviolet microbeam and found that these irradiation altered the subsequent sex-chromosome movements. Two effects were observed. In one, one of the sex chromosomes did not move at all; the sex cin after the autosomal half-bivalents have begun to move. We irradiated portions of single autosomal spindle fibres with an ultraviolet microbeam and found that these irradiation altered the subsequent sex-chromosome movements. Two effects were observed. In one, one of the sex chromosomes did not move at all; the sex cin after the autosomal half-bivalents have begun to move. We irradiated portions of single autosomal spindle fibres with an ultraviolet microbeam and found that these irradiation altered the subsequent sex-chromosome movements. Two effects were observed. In one, one of the sex chromosomes did not move at all; the sex chromosome that remained at the equator would normally have moved to the pole associated with the irradiated autosomal spindle fibre. In the second, both sex chromosomes moved to the same pole, always that of the non-irradiated side. These effects occurred whether or not autosomal anaphase movement was blocked by the irradiation. There was no wavelength dependence for altering sex-chromosome movements. Sex-chromosome movements were altered only when at least one sex-chromosomal spindle fibre was adjacent to the irradiated autosomal spindle fibre; when neither sex chromosome had a spindle fibre adjacent to the irradiated autosomal spindle fibres the chromosomes always moved normally. Irradiation of sex-chromosomal spindle fibres during sex-chromosomal anaphase showed short blockages of movement (usually 5–8 min), and then complete recovery. Direct irradiation of sex-chromosomal spindle fibres (without irradiating autosomal spindle fibres) when the autosomes were in anaphase but the sex chromosomes were in metaphase never caused abnormal sex-chromosome movements. These results eliminate the possibility that when we irradiated autosomal spindle fibres that were adjacent to sex-chromosomal spindle fibres the sex-chromosomal spindle fibres were irradiated inadvertently and were unable to recover from the damage. We suggest that the irradiations of autosomal spindle fibres alter a control system involved in “turning on' sex-chromosomal spindle fibre motors, rather than directly altering the motors. We suggest that interactions between spindle fibres are somehow involved in this control system.

scholarly journals The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. II. Kinetochore microtubules in non-treated spindles

1985 ◽  
Vol 79 (1) ◽  
pp. 39-65 ◽  
Author(s):  
B.B. Czaban ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
The Other ◽  
Spindle Fibre ◽  
Near Ultraviolet ◽  
Other Hand ◽  
Spindle Microtubules ◽  
Spindle Fibres

We determined the kinetic polarities of chromosomal spindle fibre microtubules in vivo: either the kinetochore or pole ends of chromosomal spindle fibres were irradiated with near-ultraviolet light to prevent depolymerization by colcemid. Irradiations began either just before or just after colcemid addition; cells were continually irradiated and continuously immersed in colcemid. Irradiations of kinetochore ends of chromosomal spindle fibres prevented depolymerization; irradiations of pole ends did not. Therefore, since colcemid acts by binding to the ‘on’ (assembly) ends of microtubules, the on ends of chromosomal spindle fibre microtubules are at the kinetochores. That is, in untreated chromosomal spindle fibres in vivo tubulin monomers add to kinetochore microtubules at the kinetochore ends. Tubulin diffused from the irradiation sites: irradiations of the cytoplasm sometimes prevented depolymerization of chromosomal spindle fibres. Prevention of chromosomal spindle fibre depolymerization was dependent on the distance of the irradiated region from the nearest chromosome; the longer the distance the less likely was it that the irradiation prevented depolymerization. On the other hand, prevention of chromosomal spindle fibre depolymerization was not dependent on the distance from the irradiated spot to the nearer pole. This analysis, too, we argue, strongly suggests that the kinetochore ends of the chromosomal spindle fibres are the on ends.

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