Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.

Satoru Yokawa,Tadahide Furuno,Satoshi Inouye,Takahiro Suzuki,Yoshikazu Inoh,Ayumi Hayashi

doi:10.3389/fcell.2018.00074

Satoru Yokawa, Tadahide Furuno + Show 4 more

Open Access

https://doi.org/10.3389/fcell.2018.00074

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Abstract

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

Highlights

IntroductionMast cells secrete prestored allergic mediators (e.g., histamine and serotonin), synthesize lipid mediators (e.g., leukotorienes and prostaglandins), and produce cytokines (e.g., tumor necrosis factor-α and interleukin-4)
Mast cells secrete prestored allergic mediators, synthesize lipid mediators, and produce cytokines
We found that pcDNA3-preferred human codon-optimized gene of NPY (pNPY)-preferred human codon-optimized GLuc (pGLuc) maintained a one-fifth luminescence activity to pcDNA3-pGLuc (Figure 1). These results indicated that the combination of pNPY and pGLuc was efficient for the expression of neuropeptide Y (NPY)-Gaussia luciferase (GLase) reporter protein in RBL-2H3 cells

Summary

Introduction

Mast cells secrete prestored allergic mediators (e.g., histamine and serotonin), synthesize lipid mediators (e.g., leukotorienes and prostaglandins), and produce cytokines (e.g., tumor necrosis factor-α and interleukin-4). An atomic force microscope was used to observe the morphological changes in mast cells during the degranulation process (Nakamura and Nakanishi, 1999, 2000), and several methods have been examined for detecting degranulation in living mast cells using fluorescence microscopy One such method is to stain the intracellular granules with a basic fluorescent probe, such as quinacrine and acridine orange (Kawasaki et al, 1991; Tamura et al, 2006); this stain is not highly selective because it accumulates in acidic organelles. The fluorescent avidin, which binds to negatively charged proteoglycans in the granules, was used to detect the discharged granules on the cell surface (Joulia et al, 2015) Using these methods, fluorescence signals are accumulated on the plasma membrane in accordance with the degranulation process; quantification and specificity to detect degranulation remain restricted

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