Sea urchin spermatozoa, eggs, and embryos were labeled with the universal antibody against the intermediate filament proteins (anti‐IFA) described by Pruss et al. [Cell 27:419–428, 1981] and with anti‐beta‐tubulin. Localization of these antibodies was by indirect immunofluorescence microscopy. Cytoskeleton of unfertilized eggs, prepared according to a procedure adapted from Kane [Exp. Cell Res. 162:495–506, 1986] or as described by Dufresne et al. [Biochem. Cell Biol. 66:780–791, 1988], and reacted with the anti‐IFA demonstrate a uniformly stained background except for the nuclear areas, which appear as dark rings. During the first cell cycle, the anti‐IFA staining pattern coincides with that of spindle‐associated tubulin but not with the cortical pattern of microtubules. Swimming embryos reacted with the anti‐IFA show a labeling located on the cilia and within the cytoplasm of each individual cell of the larva. In spermatozoa, the labeling occurs all along the flagellae. Immunoblots of proteins from eggs and embryos reveal one major protein of 117 kDa and sometimes a component of 66 kDa, both of which cosediment with tubulin during the isolation procedure of microtubules described by Vallee and Bloom [Proc. Natl. Acad. Sci. USA 80:6259–6263, 1983]. These data show that proteins homologous to the intermediate filament proteins reported in vertebrate cells are present in both gametes of sea urchins. The specific localization ofthese proteins in the spindle, the flagella, and the cilia suggest that they may play a significant role in the organization and function of the microtubular lattice of the spermatozoa and of the embryo.
The effects of 6-dimethylaminopurine (6-DMAP) (a putative phosphorylation inhibitor) on the state of assembly of microtubules and intermediate filaments have been studied during the first cell cycle of the sea urchin Strongylocentrotus droebachiensis. Changes in the spatial organization of cytoskeletal structures were studied by indirect immunofluorescence with anti-tubulin and anti-IFa antibodies. The rates and patterns of protein phosphorylation in control and treated eggs were also investigated. The transfer of fertilized eggs to 600 microM 6-DMAP within 4 min following insemination inhibits pronuclear migration and syngamy. This also prevents male pronuclear decondensation, while chromatin condensation and nuclear envelope breakdown do not occur in the female pronucleus. Immunolabeling with anti-tubulin antibodies reveals the presence of cortical microtubules as early as 15 min after fertilization in both control and treated eggs. However, no sperm astral microtubules could be detected in the treated eggs. At later stages, from syngamy (40 min) up to nuclear envelope breakdown (90 min), 6-DMAP affects neither cortical microtubule organization nor the state of chromatin condensation but it precludes nuclear envelope breakdown and entry into mitosis. Treatment of the fertilized eggs after nuclear envelope breakdown induces permanent chromosome decondensation and premature disappearance of the mitotic apparatus. This last event involves disruption of the spatial organization of both microtubules and putative intermediate filaments. Quantitative measurements of protein phosphorylation show that 6-DMAP efficiently and reversibly inhibits 32P incorporation into proteins. Qualitative analysis of the autoradiograms of 32P-labeled proteins separated by SDS-PAGE reveals that a major protein band, migrating with an apparent molecular weight of 31 × 10(3)Mr, is specifically dephosphorylated in eggs treated with 6-DMAP. This study suggests that protein phosphorylation is required for sperm aster microtubule growth and migration, but not for cortical microtubule polymerization. It also strengthens the hypothesis that, in sea urchin eggs, putative intermediate filaments are tightly associated with spindle microtubules. Finally, it confirms that inhibiting protein phosphorylation before nuclear envelope breakdown reversibly prevents the entry into mitosis.
The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 microM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase. To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 microM 6-DMAP and with 200 microCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly.
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