EFFECTS OF 6-DIMETHYLAMINOPURINE ON THE LENGTH OF THE CELL-CYCLE AND ON THE STATE OF PHOSPHORYLATION OF PUTATIVE INTERMEDIATE FILAMENT PROTEINS IN SEA-URCHIN EMBRYOS

The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cel l cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embr yos were transferred into sea water containing 600 mu M 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The ef fects of 6-DMAP on cell cycle length were studied by determining the t ime required for completion of mitosis upon return of the embryos in n ormal 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 th e embryos incubated for 90 minutes compared to the 30 min incubation p eriod. However, embryos transferred 0.5 min Al have a longer M-phase t han those transferred 2 minutes or later after fertilization, suggesti ng that between 0.5 and 2 min after fertilization, critical phosphoryl ating events occur which affect the commitment of the cells to enter M -phase. To study the pattern of p117 phosphorylation during the cell c ycle, the eggs were transferred 2 minutes after fertilization in prese nce of 600 mu M 6-DMAP and with 200 mu Ci/ml of P-32-orthophosphate. A nalyses of P-32-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 great ly increases at the time of pronuclear fusion, and then declines sligh tly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a p remature breakdown of the mitotic apparatus. A causal link is suggeste d between the level of phosphorylation of p117 and its state of assemb ly. (C) 1994 Wiley-Liss, Inc.