Real-time imaging of mast cell degranulation in vitro and in vivo.
Mast cells undergo degranulation in response to various stimuli and rapidly release pre-formed mediators present in secretory granules, leading to immediate-type allergic reactions. Mast cell degranulation is commonly detected and quantified in vitro by measuring histamine or β-hexosaminidase released to culture medium. However, this type of assay cannot monitor degranulation of individual cells in real time, and it is not suitable for in vivo detection of degranulation. At the aim of real time imaging of mast cell degranulation at single cell level, we here developed a fluorescent protein-based indicator of degranulation, designated immuno-pHluorin (impH). When expressed in mast cells, impH is located in the membrane of secretory granules and non-fluorescent under homeostatic conditions while it turns fluorescent following degranulation, due to the pH change inside of granules during exocytosis. impH enabled us to detect polarized degranulation within one single cell when mast cells were stimulated via the small area of cell surface. Transplantation of impH-expressing mast cells into mast cell-deficient mice demonstrated that impH could function as a real-time indicator of degranulation in vivo. Thus, impH is a useful tool for imaging of mast cell activation and degranulation in vitro and in vivo, and may be applied for screening of reagents regulating mast cell degranulation.
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Kayo
K
Horiguchi
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan. Electronic address: yoshisou.mbch@tmd.ac.jp.
Soichiro
S
Yoshikawa
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Asuka
A
Saito
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Salma
S
Haddad
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Takuya
T
Ohta
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Kensuke
K
Miyake
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Yoshinori
Y
Yamanishi
Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan.
Hajime
H
Karasuyama
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D015550:Cell Degranulation; D003470:Culture Media; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006632:Histamine / Q000737:chemistry; D006636:Histamine Release; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry; D008407:Mast Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D050683:R-SNARE Proteins / Q000737:chemistry; D022142:Secretory Vesicles / Q000378:metabolism; D013997:Time Factors; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry; D000097573:Red Fluorescent Protein
D003470:Culture Media; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006632:Histamine; D008164:Luminescent Proteins; D050683:R-SNARE Proteins; D001619:beta-N-Acetylhexosaminidases; D000097573:Red Fluorescent Protein; C485187:PHluorin; C112938:VAMP8 protein, human
Degranulation; Exocytosis; Imaging; Mast cell; VAMP-8; pH sensor