Reversal by light of the action of n-methyl n-desacetyl colchicine on mitosis
TL;DR: The N-methyl derivative of N-desacetyl colchicine (Ciba's trade name is Colcemid) was used in this study in preference to col chicine because of its effectiveness at lower external concentrations and because of fewer irreversible effects.
Abstract: Colchicine, in low concentrations, inhibits cell division in a wide variety of cell types through an effect which results in dissolution of the spindle (1, p . 62 et seq .) This is especially clearly shown in the work of Inoue (2) . When exposed to sunlight, colchicine undergoes a photochemical rearrangement to give a, s, and y lumicolchicines (3, 4) (Fig . 1) . There is also evidence that exposure to sunlight and air reduces the biological activity of colchicine (1, p. 374) . This suggests the possibility of photochemically reversing colchicine effects in living cells . The N-methyl derivative of N-desacetyl colchicine (Ciba's trade name is Colcemid) was used in this study in preference to colchicine because of its effectiveness at lower external concentrations and because of fewer irreversible effects .
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TL;DR: Although the ability of the mitotic apparatus to establish furrows can be diminished or abolished by measures that reduce its overall size, there are no decisive data concerning which of its ultrastructural components play essential roles in cytokinesis.
Abstract: The division mechanism is fixed in the surface during anaphase or about 4 minutes before furrowing begins in cylindrical cells Under experimental conditions, the minimum time that the mitotic apparatus must act upon the surface is about 1 minute The stimulus period is followed by a latent period of 2-3 minutes The time of furrow formation can be advanced or delayed by manipulating the surface and the mitotic apparatus Since furrows can be elicited long after normal division would have been completed, it is suggested that the brevity of the normal interaction period is not a consequence of the constitution of the interactants The component of the mitotic apparatus that establishes the furrow moves from the region of the mitotic axis to the surface at 6-8 microns/minute, The components of the mitotic apparatus that are essential for furrow establishment are confined to the achromatic regions In spherical cells with large asters, the spindles are not required, although the spindle's ability to establish furrows in spherical cells can be demonstrated by changing the cell's geometry In nonspherical cells with small asters, the spindle is probably the normal active agent Although the ability of the mitotic apparatus to establish furrows can be diminished or abolished by measures that reduce its overall size, there are no decisive data concerning which of its ultrastructural components play essential roles in cytokinesis The effect of changing the geometrical relation between the mitotic apparatus and the surface differs according to the region affected Division can be blocked or impeded only by changing the relation between the equatorial surface and the mitotic apparatus The ability of the mitotic apparatus to establish furrows is diminished by increasing the distance between the astral centers and also by increasing the distance between the mitotic axis and the equatorial surface The cleavage block that results from reduction in size of the mitotic apparatus can be reversed only by decreasing the distance from the mitotic axis to the equatorial surface Artificial constrictions imposed in other regions are ineffective The normal distance relation between the astral centers and the equatorial and polar surfaces in spherical eggs is not required for division Cleavage can occur when the dimensional relations are reversed Both the surface and the mitotic apparatus can interact to establish furrows after exposure to measures that disrupt their normal organization Single, isolated asters can cause furrow-like constrictions Their immediate effect is to cause local contraction in nearby surface(ABSTRACT TRUNCATED AT 400 WORDS)
370 citations
TL;DR: These two basic types of microtubule-dependent nuclear positioning may cooperate in many proliferating animal cells to achieve proper nuclear positioning.
Abstract: The mechanisms underlying two types of microtubule-dependent nuclear positioning are discussed. 'MTOC-dependent nuclear positioning' occurs when a nucleus is tightly associated with a microtubule organizing center (MTOC). 'Nuclear tracking along microtubules' is analogous to the motor-driven motility of other organelles and occurs when the nucleus lacks an associated MTOC. These two basic types of microtubule-dependent nuclear positioning may cooperate in many proliferating animal cells to achieve proper nuclear positioning. Microtubule polymerization and dynamics, motor proteins, MAPs and specialized sites such as cortical anchors function to control nuclear movements within cells.
318 citations
Cites background from "Reversal by light of the action of ..."
...3) (Hamaguchi and Hiramoto, 1986), which capitalized on the fact that colcemid, a drug which affects microtubule polymerization, can be inactivated by UV light (Aronson and Inoue, 1970)....
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...3) (Hamaguchi a Hiramoto, 1986), which capitalized on the fact that colcem a drug which affects microtubule polymerization, can inactivated by UV light (Aronson and Inoue, 1970)....
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TL;DR: Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tub insulin diffusion in the embryo cytoplasm.
Abstract: At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1-5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.
141 citations
TL;DR: It is suggested that the female pronucleus migrates to the sperm aster by attractive force between them after they were connected by astral rays in the Colcemid‐UV method.
Abstract: The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid-UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV-irradiated region during its formation. When the UV-irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV-irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.
124 citations
TL;DR: This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.
Abstract: Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.
113 citations
Cites background from "Reversal by light of the action of ..."
...This photochemically inactivates the drug and allows the eggs to assemble microtubules if they are competent to do so (3, 11, 68)....
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