Abstract
Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.
Highlights
Lysosome-Related Organelles (LROs) are functionally diverse cell type-specific subcellular compartments that share many features with lysosomes and are capable of regulated secretion in response to appropriate stimuli [1, 2]
Since the presence of adaptor protein 3 (AP-3) has not been previously reported in Mast cell (MC), the expression of adaptor protein (AP)-3 in rat peritoneal mast cells was investigated by direct immunofluorescence
The present investigation demonstrates, for the first time, that the AP-3 adaptor complex is present in rat MCs and that it is associated with both the biosynthetic and endocytic pathways of RBL-2H3 MCs
Summary
Lysosome-Related Organelles (LROs) are functionally diverse cell type-specific subcellular compartments that share many features with lysosomes and are capable of regulated secretion in response to appropriate stimuli [1, 2]. AP-3 plays a crucial role in RBL-2H3 mast cell mediator release. Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clınicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA, www.faepa.br). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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