Abstract
Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.
Highlights
The process of mast cell degranulation requires a thorough understanding of the morphological processes underlying this complex process
There are currently two competing models for degranulation: anaphylactic degranulation in which the fusion of secretory vesicles leads to formation of secretory channels and piecemeal exocytosis in which small granules bud off the larger granules and individually fuse with the plasma membrane to release their contents
Anti-dinitrophenyl (DNP) IgE-sensitized rat basophil leukemia (RBL) cells grown on gold grids were stimulated by antigen [DNP-bovine serum albumin (BSA)] via FcεRI-mediated activation, rapidly frozen, and subsequently imaged using correlative cry-fluorescence and soft X-ray tomography (SXT) (Fig. 1A,B) to determine the location of granules (Fig. 1C), followed by SXT (Fig. 1D)
Summary
The process of mast cell degranulation requires a thorough understanding of the morphological processes underlying this complex process. We employed SXT to study organelles of activated rat basophil leukemia (RBL) cells, a model for mast cells[8], at near-native morphology. Anti-dinitrophenyl (DNP) IgE-sensitized RBL cells grown on gold grids were stimulated by antigen [DNP-bovine serum albumin (BSA)] via FcεRI-mediated activation, rapidly frozen, and subsequently imaged using correlative cry-fluorescence and SXT (Fig. 1A,B) to determine the location of granules (Fig. 1C), followed by SXT (Fig. 1D).
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