Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X ray tomography

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which numerous 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 become larger and more numerous as mast cells become 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 we 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 leve