The role of spindle microtubules in the timing of the cell cycle in echinoderm eggs

Abstract

Spindle microtubules play an important role in the mechanisms that control the timing of cell cycle events in the eggs of the sea urchins L. variegatus and L. pictus. However, recent work which used colchicine to block microtubule assembly in the eggs of two other echinoderms, S. purpuratus and D. excentricus, has raised serious questions about the generality of this role for spindle microtubules. Thus, we have systematically examined the role of spindle microtubules in the timing of the cell cycle in the fertilized eggs of these latter species. We treated eggs of both species with 5-10 microM Colcemid for several minutes starting 30 min after fertilization to completely prevent spindle microtubule assembly for several h. We used Colcemid, instead of colchicine, because it is effective at lower doses and, at these doses, shows no detectable toxic side effects. We compared for control and treated eggs the time course of nuclear envelope breakdown/reformation and DNA synthesis. We found for both species that the eggs continue to cycle without spindle microtubules; mitosis is up to twice the normal duration while interphase remains essentially unaffected. To test for the possible toxic side effects of the 1-2 mM colchicine used earlier on S. purpuratus and D. excentricus, we treated eggs of these two species, and also those of L. variegatus, with 1 mM lumi-colchicine. This photo-inactivated form of colchicine, which does not bind to tubulin, substantially prolongs mitosis and, to a lesser extent, interphase. Thus, the results of the earlier work are most easily explained by the combination of specific and nonspecific effects of the 1-2 mM colchicine used. Our present results indicate that the importance of spindle microtubules in the mechanisms that control the timing of the mitosis portion of the cell cycle is a general phenomenon.

Developmental Timing in Fertilized Merogones of the Sand Dollar, Clypeaster japonicus

“…Previous observations (1,18,20), however, have shown that Colcemid, a microtubule inhibitor, does not affect the time of the first NEB in zygote nuclei in whole eggs of sea urchins. To see if this is also the case in C. japonicus, we treated whole eggs with 5 p M Colcemid continuously from 20 min after fertilization.…”

“…Yoneda and Schroeder (25) reported that colchicine prolonged the intermitotic phase of Dendraster excentricus, but the critical examination made by Sluder et a/. (20) indicated that the apparent prolongation of the intermitotic phase seen in colchicine-treated D. excentricus eggs was the consequence of some non-specific side-effects of colchicine, rather than its specific effect on microtubules. Now it appears certain that the prolongation of the mitotic cycle is induced by disintegration of the mitotic aster and spindle.…”

Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast.

“…Suppression of microtubule dynamics might produce unoccupied microtubule-binding sites at the kinetochores or affect the tension exerted by microtubules on the kinetochore, whereas failures in spindle pole duplication will prevent up to half of the kinetochores from attaching to microtubules at all. Experiments in sea urchin eggs suggest that other mechanisms of detecting spindle defects must exist since although these cells cannot delay in mitosis in response to unattached chromosomes (Sluder et al, 1994), they can still respond to spindle depolymerization (Sluder et al, 1986).…”

Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

“…Galtase-GFP-expressing embryos were exposed to nocodazole (1 M) during the interphase before the seventh cleavage. Previous studies have reported that microtubule-depolymerizing drugs prevent chromosome separation and cytokinesis while slowing down the DNA and centrosomal replication cycles (Sluder et al, 1986). The blastomeres underwent NEBD but did not form a mitotic spindle.…”