Liposits Z, Merchenthaler I, Wetsel W C, Reid J J, Mellon P L, Weiner R I, Negro-Vilar A
Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Endocrinology. 1991 Sep;129(3):1575-83. doi: 10.1210/endo-129-3-1575.
An immortalized LHRH cell line has recently been developed by genetically targeting these neurons for tumorigenesis. One of the subclones, the GT1-7 cells, was characterized at both the light and electron microscopic levels to study the cellular and subcellular organization of these cells, particularly as they relate to biosynthesis, processing, and secretion. The cells were fixed onto slides 18-36 h after plating. LHRH and GnRH-associated peptide (GAP) immunoreactivities (IR) were detected by immunocytochemistry using colloidal gold labeling. These cultured cells exhibited the classical neuronal appearance of LHRH neurons, and they established numerous interconnections. Neighboring neurons were coupled by tight junctions, while more distant cells were interconnected with neural axon-like processes and collaterals. This cellular organization is suggestive of a neural network where neuronal activity is coordinated. At the ultrastructural level, the nondividing cells possessed indented nuclei, well developed Golgi complexes, and abundant numbers of ribosomes and secretory granules. Clathrin-coated vesicles were found in fusion with the plasma membrane. The ribosomes and secretory vesicles were particularly prominent, suggestive of high rates of protein biosynthesis and secretion. All of the cells immunostained for both LHRH and GAP; however, GAP IR was always more pronounced than that for LHRH. This finding was corroborated by biochemical data reported in a companion paper. The GAP IR was associated with ribosomes and secretory vesicles. By comparison, LHRH IR was restricted mainly to the secretory vesicles. Using colloidal gold particles of different sizes to denote LHRH or GAP IR, it was determined that both GAP and LHRH IR were colocalized within the same secretory vesicle. Taken together, these data suggest that pro-LHRH is biosynthesized on the ribosomes, packaged as an intact protein into the secretory vesicles, processed to LHRH and GAP-(1-56) within these vesicles, and transported to the periphery of the cell in preparation for secretion. These morphological data emphasize the utility of using these immortalized LHRH neuronal cells to dissect the cellular and subcellular architecture involved in biosynthesis, processing, and secretion. In addition, our results provide the first detailed evidence for the intracellular pathway involved in pro-LHRH biosynthesis, processing, and secretion in these cultured neuronal cells.
最近通过基因靶向这些神经元进行肿瘤发生，开发出了一种永生化的促性腺激素释放激素（LHRH）细胞系。其中一个亚克隆，即GT1-7细胞，在光学和电子显微镜水平上进行了表征，以研究这些细胞的细胞和亚细胞组织，特别是它们与生物合成、加工和分泌的关系。细胞在接种后18 - 36小时固定在载玻片上。使用胶体金标记通过免疫细胞化学检测LHRH和促性腺激素释放激素相关肽（GAP）免疫反应性（IR）。这些培养的细胞呈现出LHRH神经元的典型神经元外观，并且它们建立了许多相互连接。相邻神经元通过紧密连接相连，而距离较远的细胞则通过神经轴突样突起和侧支相互连接。这种细胞组织提示了一个协调神经元活动的神经网络。在超微结构水平上，不分裂的细胞具有凹陷的细胞核、发达的高尔基体复合物以及大量的核糖体和分泌颗粒。发现网格蛋白包被的小泡与质膜融合。核糖体和分泌小泡特别突出，提示蛋白质生物合成和分泌的速率很高。所有细胞对LHRH和GAP均呈免疫染色阳性；然而，GAP IR总是比LHRH更明显。一篇配套论文中报道的生化数据证实了这一发现。GAP IR与核糖体和分泌小泡相关。相比之下，LHRH IR主要局限于分泌小泡。使用不同大小的胶体金颗粒来表示LHRH或GAP IR，确定GAP和LHRH IR共定位在同一个分泌小泡内。综上所述，这些数据表明前体LHRH在核糖体上生物合成，作为完整蛋白质包装到分泌小泡中，在这些小泡内加工成LHRH和GAP-(1-56)，并运输到细胞周边准备分泌。这些形态学数据强调了使用这些永生化LHRH神经元细胞来剖析生物合成、加工和分泌所涉及的细胞和亚细胞结构的实用性。此外，我们的结果为这些培养的神经元细胞中前体LHRH生物合成、加工和分泌所涉及的细胞内途径提供了首个详细证据。
