Cultured cells derived from micromeres isolated from sea urchin embryos at the 16 cell stage are known to show outgrowth of pseudopodial cables followed by spicule rod formation when cultured in the presence of horse serum. Micromere-derived cells cultured with bovine insulin showed pseudopodial cable growth but did not produce spicule rods. Micromere-derived cells reversibly bound to insulin through out the period between 3 and 20 hr of culture. The dissociation constant of insulin with these cells was about 5.1 x 1 O-'OM during the whole culture period examined. Horse serum, as well as blastocoelic fluid obtained from early gastrulae, concentration-dependently reduced the amount of insulin bound to these cells, but the bound insulin was scarcely replaced by any proteins tested, such as bovine serum albumin. The micromerederived cells were bound to have an insulin-binding protein, that may be the receptor for insulin or insulin-like proteins. The insulin-binding protein had a smaller molecular weight than the insulin receptor of mammalian cells. The binding of insulin with this protein in micromere-derived cells probably results in pseudopodial cable growth.
In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of 32P incorporation into protein (protein phosphorylation) followed by activation of 32P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds. In cells treated with horse serum treated cells, high rates of protein phosphorylation and RNA synthesis were maintained even after the initiation of cable growth and about 5 h later, spicule rods were produced.In cells treated with horse serum, actinomycin D treatment started at the time of initiation of cable growth, cables were formed but formation of spicule rods was blocked. These results suggest that horse serum contains some other substance besides insulin-like ones, which induces expression of genes that are indispensable for spicule rod formation.Insulin treatment did not induce spicule rod formation.
In cultured cells derived from isolated micromeres of 16‐cell stage sea urchin embryos, which undergo insulin‐induced pseudopodial cable growth, specific and reversible insulin binding by a 52‐kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin‐binding capacity in micromere‐derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U‐3H]uridine incorporation into RNA and [35S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres. The capacity in the plasma membrane fraction increased, accompanied by its decrease in the microsome fraction, during 5 h of culture of micromere‐derived cells. The insulin receptor is probably accumulated in microsomes of presumptive micromeres prior to the 16‐cell stage and transferred to the plasma membrane, resulting in an increase in the insulin binding capacity of micromere‐derived cells during 5 h of culture.
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