Malosio Maria Luisa, Giordano Tiziana, Laslop Andrea, Meldolesi Jacopo
Department of Neuroscience, DIBIT, Vita-Salute San Raffaele University, Via Olgettina, 58, 20132 Milan, Italy.
J Cell Sci. 2004 Feb 15;117(Pt 5):743-9. doi: 10.1242/jcs.00934. Epub 2004 Jan 20.
Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.
致密核心颗粒是一种典型的胞吐细胞器，人们普遍认为其表达受神经元和神经分泌细胞特有的协同基因表达机制控制。然而，最近对PC12细胞的研究表明，颗粒/细胞的数量仅取决于其一种货物蛋白嗜铬粒蛋白A的水平，嗜铬粒蛋白A通过一种开/关切换机制调节其他蛋白的代谢，进而调节细胞器的组成。此外，据报道，转染嗜铬粒蛋白A可诱导CV-1细胞系的非神经分泌成纤维细胞中出现致密核心颗粒。在此，我们使用的不是异质性的PC12细胞系，而是从中分离出的几个克隆，重新研究了嗜铬粒蛋白A的作用。在这些克隆中，无论是直接研究还是转染嗜铬粒蛋白A反义序列后进行研究，嗜铬粒蛋白A与其分泌蛋白伴侣嗜铬粒蛋白B之间的比例都不是恒定的，而是高度可变且明显随机变化。通过共聚焦免疫荧光观察，在单个克隆和亚克隆的细胞之间以及单个细胞的颗粒之间，也能看到嗜铬粒蛋白A/嗜铬粒蛋白B比例的变异性。此外，在一个致密核心颗粒数量较少（为参考克隆的五分之一）的PC12克隆中，稳定和瞬时转染嗜铬粒蛋白A均未能显著改变细胞器的数量，尽管颗粒蛋白增加了几倍。最后，在三种非神经分泌细胞（CV-1、腺癌TS/A和一个无分泌能力的PC12克隆）中，转染的嗜铬粒蛋白A大多积聚在高尔基体/反式高尔基体区域，并在环己酰亚胺处理期间的免疫荧光和脉冲追踪实验中显示，从静息细胞中迅速释放（组成型分泌）。只有一小部分被分选到离散的细胞器中，这些细胞器不是致密核心颗粒，而是主要为溶酶体，因为它们不含嗜铬粒蛋白B，并且对内体-溶酶体晚期标记物lamp1和lamp（此处原文可能有误，推测为lamp3）大多呈阳性。因此，致密核心颗粒是神经元和神经分泌细胞的真正标志。它们的数量/细胞似乎与嗜铬粒蛋白A无关，其组成似乎也不恒定；特别是，它们在嗜铬粒蛋白A/嗜铬粒蛋白B比例上表现出相当大且迄今无法解释的变异性。
