Institut National de la Santé et de la Recherche Médicale (Inserm) U982, University of Rouen, Mont-Saint-Aignan 76821, France.
Endocrinology. 2012 Sep;153(9):4444-56. doi: 10.1210/en.2012-1436. Epub 2012 Jul 31.
Chromogranins are a family of acidic glycoproteins that play an active role in hormone and neuropeptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca(2+)-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography-tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin- and/or Ca(2+)-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca(2+)-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca(2+)-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.
嗜铬粒蛋白是一组酸性糖蛋白，它们在神经内分泌细胞的分泌颗粒生物发生中发挥关键作用，从而积极参与激素和神经肽的分泌。然而，其颗粒生成活性的分子机制仍不完全清楚。因为我们之前证明了主要分泌颗粒成分嗜铬粒蛋白 A（CgA）的表达能够诱导非内分泌 COS-7 细胞中分泌颗粒的形成，所以我们决定使用该模型来剖析 CgA 引发的导致这些颗粒生物发生和转运的机制。通过定量活细胞成像，我们首先表明 CgA 诱导的细胞器表现出 Ca2+依赖性转运，与天然的含有病毒 G 蛋白的囊泡性口炎病毒组成型囊泡相反。为了鉴定赋予新形成的颗粒这些特性的蛋白质，我们开发了 CgA 稳定表达的 COS-7 细胞，通过亚细胞分级分离纯化其含有 CgA 的颗粒，并通过液相色谱-串联质谱法分析颗粒蛋白质组。该分析揭示了几种胞质蛋白与颗粒膜的结合，包括 GTPases、基于细胞骨架的分子马达以及具有肌动蛋白和/或 Ca2+结合特性的其他蛋白质。此外，细胞骨架的破坏不仅影响 CgA 含颗粒的分布和运输，还影响 Ca2+引发的其内容物的胞吐作用，表明这些颗粒与微管和皮质肌动蛋白相互作用，以调节其内容物的释放。这些数据首次表明，神经内分泌因子 CgA 诱导细胞骨架、GTP 和 Ca2+结合蛋白在持续分泌的 COS-7 细胞中的募集，以产生具有神经内分泌分泌颗粒典型动力学和胞吐特性的囊泡。
