Chromogranin A Induces the Biogenesis of Granules with Calcium- and Actin-Dependent Dynamics and Exocytosis in Constitutively Secreting Cells

Abstract

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 Ca2+-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 Ca2+-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca2+-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 Ca2+-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.