Cell Differentiation, 11 (1982) 357--358 357 Elsevier/North-Holland Scientific Publishers, Ltd.

EFFECTS OF CAFFEINE ON MITOSIS IN EGGS OF THE SEA URCHIN STRONGYLOCENTROTUS PURPURATUS: THE POSSIBLE ROLE OF CALCIUM

PATRICIA HARRIS

Department of Biology, University of Oregon, Eugene, OR 97403, U.S.A.

tubulin spindle pacemaker sea urchin

There is a great deal of indirect evidence that intracellular calcium regulation plays an important role in the formation and break- down of the mitotic apparatus, in chromo- some movement, and possibly even in the timing of the mitotic cycle. In sea urchin eggs, fine structure studies show a large mass of membrane-bounded vesicles associated with the mitotic apparatus (Harris, 1975), and these have recently been shown to sequester calcium in a manner similar to the sarcoplasmic reti- culum (Silver et al., 1980). Furthermore, a calcium-binding relaxing factor has been local- ized in dividing sea urchin eggs by means of fluorescent antibodies (Kinoshita and Yazaki, 1967). It was of interest, therefore, to s tudy the effects on mitosis of a drug that is known to affect intracellular calcium regulation. Caffeine was chosen because it delays or blocks mitosis in various cell types and, at least in the short term, is reversible. It is known to cause a release of calcium from the sarcoplasmic reticulum (Weber and Herz, 1968), and in sea urchin eggs it inhibits a Ca- ATPase associated with calcium sequestering, probably by its effect on the sulfhydryl- disulfide balance in the cell (Nath and Reb- hun, 1976). The antimitotic effects of the caffeine are not due to an increase in cyclic AMP resulting from inhibition of phospho- diesterase, and cyclic AMP has been shown to have no effect on mitosis in sea urchin eggs (Nath and Rebhun, 1976). Earlier studies have shown that caffeine causes shrinking of

the mitotic spindle in sea urchin eggs at meta- phase. The object of this s tudy was to examine in more detail the structural changes during spindle breakdown and recovery when re- turned to sea water, and to fol low the long- term effects of the drug on blocked cells.

Fertilized eggs of the sea urchin Strongyl- ocentrotus purpuratus were treated at prome- taphase with 10 mM caffeine in normal sea water, the lowest concentrat ion of caffeine which blocked mitosis. Samples were taken at 15--30 min intervals over a period of several hours for tubulin localization by indirect im- munofluorescence microscopy, for phase- contrast studies of glutaraldehyde-fixed whole mounts, and for observation of osmium-fixed sections for light and electron microscopy. Photographic recording was also made of living cells.

Within 15 min of addition of caffeine the spindle had shortened until both mitotic centers and the chromosomes were grouped together at the center of the metaphase plate and no microtubules could be detected by tubulin immunofluorescence labeling. The eggs soon recovered their ability to assemble micro- tubules and by 30 min a monaster began to form around the centers. As the monaster grew to fill the entire cell, the chromosomes decondensed to form an interphase nucleus. During the stage equivalent to telophase, furrows a t tempted to form. Some cells re- tained the partial furrows, bu t most cells rounded up again until the next cycle. Break-

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down of the monaster then proceeded from the cell center to the periphery, as previously observed in asters of normal divisions. This was soon followed by the formation of new asters, Chromosome condensation, nuclear membrane breakdown, and the formation of another large monaster. The cycle period varied from one batch of eggs to another, but was roughly two to three times longer than the normal second division. This cycling continued in surviving cells for at least 24 h. Fig. 1 shows the sequence of stages up to 240 min after addition of caffeine.

The rapid caffeine-induced breakdown of the mitotic apparatus, followed by cyclic formation and breakdown of monasters, is consistent with the idea that caffeine causes a release of calcium within the cell, which is then periodically resequestered and released. Such caffeine-induced calcium oscillations have been demonstrated previously in skinned muscle preparations (Endo et al., 1970) and could provide a model for the timing mecha- nism for monaster cycling as well as for early cleavage divisions. The increase in period length of the monaster cycles over the normal div- ision time may reflect the partial inhibition of the Ca-ATPase of the calcium pump.

The pattern of aster formation and break- down, always proceeding from the aster center toward the cell periphery, suggests that some- thing in the aster centers may serve as a pace- maker initiating a wave of microtubule break- down. If calcium is a factor in the aster break-

0

150 IBO 240

TfME IN MfNUTES AFTER ADDITION OF CAFFEINE

Fig. 1. Pattern of first two monaster cycles following addition of 10 mM caffeine at prometaphase.

down it appears to be released as a regenerative wave similar to the fertilization reaction i ~ Medaka eggs (Gilkey et al., 1978) rather than simultaneously throughout the cell.

If prometaphase-blocked eggs are allowed to recover in normal sea water after 15 min in caffeine, they usually form a monaster or asymmetric division because the centers have not had time to separate sufficiently. How- ever, if the eggs are allowed to recover after nuclei have reformed, i.e. at a different phase of the cycle, the resulting division is directly from one to four cells. Caffeine apparently affects the ability of the organizing centers to separate and form effective poles, and in this respect the caffeine-induced monaster cycling is similar to that occurring in non-dividing artificially activated eggs. I t is not known whether the effect on the organizing centers is mediated through the calcium-regulating system or is a direct effect of caffeine on the centrioles or organizing centers themselves.

The results of the experiments with caffeine on dividing sea urchin eggs provide further in- direct evidence that calcium is important in regulating the events of mitosis and possibly timing the mitotic cycle itself. However, direct proof awaits reliable measurements of intra- cellular free calcium throughout the division cycle and its localization within the cell.

References

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458 (1967). Nath, J. and L.I. Rebhun- J. Cell Biol. 68, 440--

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