DeSimone Douglas W, Spiegel Melvin
Department of Biological Sciences, Dartmouth College, 03755, Hanover, NH, USA.
Rouxs Arch Dev Biol. 1986 Sep;195(7):433-444. doi: 10.1007/BF00375747.
The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.
基于凝集素结合对刺冠海胆(Lytechinus pictus)和斑点海胆(Arbacia punctulata)胚胎的基底膜和内部细胞外基质进行了表征。在显微注射刀豆球蛋白A-异硫氰酸荧光素(Con A-FITC)和小麦胚凝集素-异硫氰酸荧光素(WGA-FITC)后,观察到凝集素结合的发育阶段特异性模式。对照、无硫酸盐海水(SFSW)和衣霉素处理的胚胎之间,凝集素特异性模式有所不同。注射Con A导致上皮细胞变圆,在衣霉素存在下培养的胚胎中最为明显。通过Triton X-100提取整个胚胎来分离基底膜。蛋白质通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,电泳转移到硝酸纤维素膜上,然后与生物素化凝集素一起孵育。用抗生物素蛋白过氧化物酶检测凝集素结合糖蛋白。斑点海胆早期发育阶段Con A结合蛋白的电泳模式与对照和SFSW胚胎原肠胚形成时出现的几种低分子量蛋白相似。斑点海胆和刺冠海胆对照胚胎中的WGA结合仅限于一种125,000道尔顿的糖蛋白(gp125)。除了gp125,在SFSW胚胎中还检测到几种高分子量的WGA结合糖蛋白。证据表明,间充质迁移和原肠胚形成与细胞外基质的分子组成变化相关。