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Biomedical subjects

A Forer

Publications and source records attributed to A Forer.

At least 37 records · Page 2Linked to original sources
PubMed1991

Kinetochore function can be altered by ultraviolet microbeam irradiation without loss of the associated birefringent spindle fibre.

We have irradiated kinetochores of chromosomes in spermatocytes of crane flies (Nephrotoma abbreviata (Loew)) and Nephrotoma suturalis (Loew), while observing the cells using polarization microscopy. Irradiation of a kinetochore of one sex chromosome with 0.106 ergs microns-2, the minimum dose needed to stop movement, had no effect on the birefringence of the irradiated kinetochore's spindle fibre. Irradiation of the kinetochore of an autosomal half-bivalent in anaphase, with the same dose, had no effect on the birefringence of the irradiated kinetochore's spindle fibre, but nonetheless the anaphase movements of all six autosomal half-bivalents were stopped, temporarily, for up to 20 min. Irradiations of the kinetochores of an autosomal half-bivalent with higher doses (0.301 ergs microns-2) caused loss of birefringence of the irradiated kinetochore's spindle fibre, and the movements of all six autosomal half-bivalents were stopped permanently. We argue that the ultraviolet microbeam differentially affects two functions of the kinetochore: (1) a 'signalling' function, and (2) microtubule attachment, with the signalling function being altered at doses lower than that of microtubule attachment.

Animals↗
PubMed1991

Electron-microscopic and immunochemical analysis of kinetochore microtubules after ultraviolet microbeam irradiation of kinetochores.

We used an ultraviolet microbeam to irradiate kinetochores of chromosomes in crane-fly spermatocytes. We used one of two doses, low (0.106 erg microns-2) or high (0.301 erg microns-2), and then studied the microtubules in those spindles using electron microscopy or immunofluorescence microscopy. After irradiation with low doses microtubules are present as usual, with normal fluorescence and in normal numbers. After irradiation with high doses microtubules are no longer associated with the irradiated kinetochore. After irradiation with either dose, non-kinetochore microtubules are in smaller numbers in the irradiated half-spindle than in the non-irradiated half-spindle or in non-irradiated cells. Since irradiation with low doses alters interchromosomal 'signals', but microtubules remain attached to the kinetochore, we argue that low doses of ultraviolet light damage a signal-related function of kinetochores without altering the ability of the kinetochores to bind microtubules.

Animals↗
PubMed1991

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

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.

Animals↗
PubMed1989

Identifying the site of microtubule polymerization during regrowth of UV-sheared kinetochore fibres using antibodies against acetylated alpha-tubulin.

Areas of reduced birefringence (ARBs) produced on chromosomal fibres of crane-fly spermatocyte spindles by ultraviolet microbeam irradiation move poleward. The ARB is due to the depolymerization of the microtubules in that area, and its poleward motion is due in part to the lengthening of that part of the kinetochore fibre which is left attached to the kinetochore after shearing the microtubules. We tested whether the lengthening of this fibre is due to the polymerization of microtubules at the growing edge of the ARB by staining growing fibres in irradiated spindles with antibodies to tubulin and to acetylated tubulin. We have previously argued that newly-polymerized kinetochore microtubules are not acetylated, whereas older kinetochore microtubules are (Wilson & Forer, 1989). Therefore we expected to see an absence of staining with antibodies to acetylated tubulin at the edge of the ARB if microtubules were polymerizing there. There is no absence of staining, however, which suggests that growth of the sheared microtubules does not occur at the ARB edge. Other possibilities are discussed.

Acetylation↗
PubMed1989

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

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.

Animals↗
PubMed1988

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

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.

Animals↗
PubMed1988

Ultraviolet microbeam irradiation of microtubules in vitro. The action spectrum for local depolymerization of marginal band microtubules in vitro matches that for reducing birefringence of chromosomal spindle fibres in vivo.

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which microtubules are locally depolymerized.

Animals↗
PubMed1987

The role of the phosphatidylinositol cycle in mitosis in sea urchin zygotes. Lithium inhibition is overcome by myo-inositol but not by other cyclitols or sugars.

We have investigated the role of the phosphatidylinositol (PI) cycle in cellular events between fertilization and first cleavage in zygotes of the sea urchin Lytechinus pictus. The effects of lithium were studied: The lithium-induced changes due to effects on the PI cycle were reversed by myo-inositol, the next step in the cycle after the lithium block, but were not reversed by scyllo-inositol or other cyclitols or sugars. In this way we implicated the PI cycle in the formation of streak birefringence, in nuclear membrane breakdown, in onset of anaphase, and in cytokinesis. With respect to karyokinesis, mitotic apparatus (MA) structure often was altered when the PI cycle was blocked, and anaphase was blocked when the PI cycle was blocked. For all stages, the effects of 400 mM lithium were overcome by 10-100 microM myo-inositol. Excess myo-inositol potentiated the effect of lithium on MA structure (and on cytokinesis), suggesting that there is a negative feedback loop in the control of the PI cycle.

Animals↗
PubMed1987

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

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.

Actins↗
PubMed1987

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

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.

Actins↗
PubMed1985

CA++ in fertilization and mitosis: the phosphatidylinositol cycle in sea urchin gametes and zygotes is involved in control of fertilization and mitosis.

We determined that the phosphatidylinositol (PI) cycles in both sea urchin sperm and eggs are necessary for normal fertilization, and that the PI cycle in sea urchin zygotes is involved in control of mitosis. The PI cycle is involved in Ca++ homeostasis so our data are direct evidence that Ca++ is involved with control of mitosis and fertilization. We implicated the PI cycle by adding Li+ to sea urchin eggs, sperm, or zygotes: those effects of Li+ due to effects on the PI cycle were overcome by myo-inositol but not by its optical isomer, scyllitol, and not by mannitol.

Animals↗
PubMed1985

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

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).

Actins↗
PubMed1985

The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. I. Kinetochore microtubules that re-form after treatment with colcemid.

In newly formed chromosomal spindle fibres we determined the kinetic polarities of the microtubules, that is, the ends to which tubulin monomers add. Spindles disappeared after cells were continuously immersed in colcemid; then portions of the cells were continuously irradiated with a microbeam of near-ultraviolet light to reverse locally the effect of the colcemid. From the following lines of evidence we conclude: that microtubules are organized by the chromosomes; and that tubulin monomers add to the chromosomal spindle fibres at the kinetochore. When chromosomes were irradiated chromosomal spindle fibres grew in different directions, not necessarily focussed to a common pole; this would not occur if the chromosomal spindle fibres were organized by poles. Chromosomal spindle fibres were sometimes associated with only some of the chromosomes; this would not occur if the fibres were organized by the poles. Thus, chromosomal spindle fibres are organized solely by chromosomes; these spindle fibres are functional since the associated chromosomes moved in anaphase. When chromosomes were irradiated the re-formed spindle fibres grew up to 10 microns past the edges of the irradiating spot. Experimentally, free tubulin did not diffuse more than 4-5 microns from the irradiated spot. Thus we conclude that the tubulin monomers add at the kinetochores and not at the distal ends of the fibres.

Animals↗
PubMed1985

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

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.

Animals↗
PubMed1984

Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. I. Description of the system and general results.

A new system, based on a video digitizer interfaced to a microcomputer, has been developed to quantify birefringence of individual chromosomal spindle fibres from videotaped images of spindles. (The system also can be used for any other purpose that requires the analysis of video intensities.) Retardations along the lengths of single chromosomal spindle fibres have been studied throughout metaphase and anaphase in cells kept at constant temperatures. The instrumental readings are accurate to within less than 0.06 nm retardation, but operationally the retardation values along a single chromosomal spindle fibre can vary by up to 0.15 nm, primarily because of variation in operator definition of the spindle fibre. Retardations vary with position along the fibre. During anaphase the retardations along a given chromosomal spindle fibre do not move poleward, but rather change as if the oriented material is disorganized at the kinetochore. The retardation at the kinetochore of a chromosomal spindle fibre does not change during anaphase, except for nonpredictable jumps of 20-30% that sometimes occur. Thus there is no 'decay of birefringence' during anaphase, such as has been described in other species. In this regard our data, that pertain only to single chromosomal spindle fibres, differ from those previously published; we argue that this is because the published data deal with mixtures of chromosomal and continuous spindle fibres, and because changes in birefringence can appear to occur, artefactually, when measurements of birefringence are made at a single spot in a spindle.

Anaphase↗
PubMed1984

Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. II. Crane-fly spermatocyte chromosomal spindle fibres are not temperature-labile.

Retardations were measured along the lengths of single chromosomal spindle fibres, from metaphase through anaphase, from video-taped images of crane-fly spermatocytes incubated at various temperatures (4-30 degrees C). These measurements were made using a video digitizer interfaced to a microcomputer. Over most of the range of temperatures at which normal anaphase movement occurs the chromosomal spindle fibres are not temperature-labile. The non-specific and continuous fibre birefringence is temperature-labile, however. The data are discussed with respect to the 'dynamic equilibrium' model of anaphase chromosome movement. We conclude that, since single chromosomal fibre birefringence is not temperature-labile over most of the range of temperatures at which normal anaphase chromosome movement occurs, these data do not support the dynamic equilibrium model of anaphase chromosome movement.

Anaphase↗
PubMed1984

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

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.

Animals↗