Studies on outgrowth of pseudopodial cables and spicule rod formation in cultured derived from micromeres of sea urchin embryos.

已知海胆胚胎的微粒体衍生细胞在与马血清或从海胆胚胎分离的囊胚腔液培养期间产生伪足索和骨针杆。在本研究中，胰岛素被发现诱导电缆的增长没有骨针杆形成。SDS-PAGE显示胰岛素处理的细胞中有胰岛素结合蛋白。胰岛素与细胞结合，Kd约为0.5 pM，结合胰岛素在马血清和囊胚腔液中被未知化合物取代。这表明马血清和囊胚腔液含有胰岛素样化合物。由于受体蛋白从微粒体易位到质膜，细胞中的胰岛素结合在6小时内增加，并且在不存在马血清或胰岛素的情况下，在培养40小时内保持高水平。在具有高胰岛素结合能力的细胞中，胰岛素以及马血清，在蛋白磷酸化活化后发生RNA合成的增加，并最终导致电缆生长。蛋白磷酸化的抑制降低了RNA合成的速率，并且在胰岛素诱导的蛋白磷酸化增强后，放线菌素D抑制RNA合成导致伪足索生长的阻断。在用马血清保存的细胞中，即使在电缆生长开始后，蛋白质磷酸化和RNA合成的速率仍然很高。放线菌素D引入后电缆的生长开始允许用马血清或胰岛素处理的细胞引起伪足电缆，但阻止骨针的形成在马血清处理的细胞。马血清和囊胚腔液似乎含有胰岛素以外的信号化合物。

Micromere-derived cells of sea urchin embryos are known to produce pseudopodial cables and spicule rods during culture with horse serum or blastocoelic fluid isolated from sea urchin embryos. In the present study, insulin was found to induce the cable growth without spicule rod formation. Insulin binding protein was found by SDS-PAGE of insulin treated cells. Insulin bound to the cells with Kd of about 0.5 pM and bound insulin was replaced by unknown compounds in horse serum and blastocoelic fluid. This indicates that horse serum and blastocoelic fluid contain insulin-like compounds. Insulin binding in the cells increased in 6 hr period due to translocation of the receptor protein from microsome to plasma membrane and was maintained high up to 40 hr of culture in the absence of horse serum or insulin. In the cells with high insulin binding capacity, insulin, as well as horse serum, augmentation of RNA synthesis occurred following activation of protein phosphorylation and finally resulted in the cable growth. Inhibition of protein phosphorylation reduced the rate of RNA synthesis and inhibition of RNA synthesis by actinomycin D after insulin-induced augmentation of protein phosphorylation resulted in blockage of pseudopodial cable grwoth. In the cells kept with horse serum, the rates of protein phosphorylation and RNA synthesis remained high even after the initiation of cable growth. Actinomycin D introduced after the cable growth initiation allowed the cells treated with horse serum or insulin to cause pseudopodial cables but blocked the spicule formation in horse serum-treated cells. Horse serum and blastocoelic fluid seems to contain signalling compounds other than insulin.