Kaether C, Salm T, Glombik M, Almers W, Gerdes H H
Department for Neurobiology, University of Heidelberg, Germany.
Eur J Cell Biol. 1997 Oct;74(2):133-42.
Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.
人嗜铬粒蛋白B（hCgB）是神经内分泌分泌颗粒的一种可溶性标记蛋白，它与绿色荧光蛋白（GFP）融合。使用具有不同折叠特性的两种GFP突变体S65T和增强型绿色荧光蛋白（EGFP）分别产生两种重组蛋白，即hCgB - GFP（S65T）和hCgB - EGFP。瞬时表达后，只有hCgB - EGFP在神经内分泌细胞系PC12中引发绿色荧光。用[35S]硫酸盐进行脉冲追踪实验，随后进行亚细胞分级分离，结果表明hCgB - EGFP被高效分选至未成熟分泌颗粒（ISG）。共聚焦显微镜显示，荧光性的hCgB - EGFP主要与突触结合蛋白共定位，突触结合蛋白是分泌颗粒和突触样微泡的膜标记物，并且与分泌颗粒的可溶性标记物内源性大鼠嗜铬粒蛋白B（rCgB）显著共定位。在用5 mM Ba2+刺激转染细胞或用50 mM K+去极化后，hCgB - EGFP发生调节性胞吐作用。通过共聚焦显微镜观察活PC12细胞质膜（PM）下方绿色荧光分泌颗粒的动态变化。这些囊泡中的大多数在8.5秒内没有移动，就好像它们停靠在那里一样。相比之下，在神经生长因子（NGF）诱导的细胞中，胞体PM下方的大多数分泌颗粒在同一时间段内移动，而在神经突中仅观察到很少的移动。这些发现表明，在分化的PC12细胞中，大多数停靠区不在胞体中，而是沿神经突分布。总之，融合蛋白hCgB - EGFP为实时研究蛋白质分泌调节途径中的囊泡运输提供了一个强大的工具。
