Erratum: Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

1987 ◽  
Vol 65 (6) ◽  
pp. 593-593
Author(s):  
Paula Wilson ◽  
Esther Fuller ◽  
Arthur Forer
Keyword(s):  
Ultraviolet Light ◽  
Ultraviolet Microbeam

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.

Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction

1987 ◽  
Vol 65 (4) ◽  
pp. 363-375 ◽  
Author(s):  
Paula Wilson ◽  
Arthur Forer
Keyword(s):  
Ultraviolet Light ◽  
Dose Response ◽  
Thin Filament ◽  
Primary Effect ◽  
Chromosome Movement ◽  
Response Curves ◽  
Rabbit Psoas ◽  
Ultraviolet Microbeam ◽  
Filament Structure ◽  
Wavelength Light

Glycerinated rabbit psoas myofibrils, F-actin, and myofibril ghosts were irradiated with ultraviolet light (UV) to investigate how UV blocks myofibril contraction. Myofibril contraction is most sensitive to 270- and 290-nm wavelength light. We irradiated I and A bands separately with 270- and 290-nm wavelength light using a UV microbeam and constructed dose-response curves for blocking sarcomere contraction. For both wavelengths, irradiations of A bands required less energy per area to block contraction than did irradiations of I bands, suggesting that the primary effects of both 270- and 290-nm wavelength light in stopping myofibril contraction are on myosin. We investigated whether the primary effect of UV in blocking I-band contraction is the depolymerization of actin by comparing the relative sensitivities of I-band contraction, F-actin depolymerization, and thin filament depolymerization to 270- and 290-nm light. We also compared the dose of UV required to depolymerize F-actin in solution with the dose needed to block I-band contraction and the dose required to alter thin filament structure in myofibril ghosts. The results confirm that UV blocks I-band contraction by depolymerizing actin. We discuss how the results might be relevant to the hypothesis that an actomyosin-based system is involved in chromosome movement.

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.

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.

scholarly journals In ultraviolet microbeam irradiations, characteristics of the monochromator and lamp affect the spectral composition of the ultraviolet light and probably the biological results

1991 ◽  
Vol 98 (3) ◽  
pp. 415-422
Author(s):  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Spectral Distribution ◽  
Spectral Composition ◽  
Band Width ◽  
Microbeam Irradiation ◽  
Ultraviolet Microbeam ◽  
Spectral Distributions

Biological conclusions recently published concerning ultraviolet (u.v.) microbeam irradiation of spindles are different from those we previously published. Several technical differences between the two sets of experiments were investigated. The spectral distributions in the light emitted from mercury-arc, xenon-mercury-arc, and xenon-arc lamps were measured, as were the spectral distributions after the light from these lamps passed through a monochromator that was set to various wavelengths and various half-band-widths. Both the source of the u.v. light and the half-band-width of the monochromator influence the spectral distribution of the light leaving the monochromator: depending on the conditions, the light leaving the monochromator is not necessarily of the same wavelength as that to which the monochromator is set. Differences in these aspects of the experiments could easily give rise to the different biological conclusions reached in the two sets of experiments.

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.

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

1987 ◽  
Vol 65 (4) ◽  
pp. 376-385 ◽  
Author(s):  
Paula Wilson ◽  
Esther Fuller ◽  
Arthur Forer
Keyword(s):  
Ultraviolet Light ◽  
Staining Pattern ◽  
Apparent Contradiction ◽  
Thin Filaments ◽  
Rhodamine Phalloidin ◽  
Fluorescence Image ◽  
Rabbit Psoas ◽  
Ultraviolet Microbeam ◽  
Presence And Absence ◽  
Over Time

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine–phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.

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