Chiang, M.-L.; Chen, H.-Y.; Lai, L.-J.; Guttmann, P.; Hsieh, C.-C.: Three-dimensional ultrastructural analysis of mast cell degranulation - Meeting Abstract. Allergy 68 (2013), p. 7-8
Mast cells are a key cell type that plays an important role in allergic reactions. They contain granules harboring mediators that are released when the cells are activated, through a process called degranulation. The granule homeostasis in mast cells has been studied in the past mainly by EM, but this approach distorts the fine structure of subcellular organelles during sample preparation. The migration and fusion of granules in mast cells degranulation have not been studied in detail in 3D. We have studied the dynamic process of degranulation, including granule fusion, migration, and mediator release by soft x-ray tomography (SXT) and super-resolution microscopy (SRM). Rat basophilic leukemia cells (RBL-2H3) were sensitized with anti-DNP IgE and then challenged with DNP-BSA for different periods. They were then frozen immediately by plunge freezing system in liquid ethane and the frozen cells were loaded onto a vacuum chamber for soft-x-ray transmission microscopy examination. The acquired images were reconstructed by IMOD software and further processed and segmented by Amira software. 3D images of fusion granules at early time points and late time points were obtained. We observed that fusing granules were surrounded by mitochondria, as demonstrated by other investigators previously. By using confocal microscopy and SRM, we found that granules migrated from perinuclear site to cell membrane and sometimes were accumulated in the polarization site of sensitized RBL-2H3 cells at late time points of degranulation. These results indicate that granule fusion may be a sequential event in that more fusion events occur closer to the plasma membrane. Our preliminary data also revealed granule fusion with the plasma membrane as well as the raffle membrane structures.