1996 Fiscal Year Final Research Report Summary
Confocal and Probe Microscopy for Studying Immune Responses
| Project/Area Number |
07457531
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
NAKANISHI Mamoru Nagoya City University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90090472)
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| Co-Investigator(Kenkyū-buntansha) |
FURUNO Tadahide Nagoya City University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80254308)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Confocal Microscopy / Basophils / Atomic force microscopy / Antigen stimulation / SH2 / CD63 / Degranulation / Imaging analysis |
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| Research Abstract |
SH2 domains are present in cytoplasmic tyrosine kinases and also in various proteins involved in signal transduction. We prepared here monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Fluorescence images observed by confocal laser microscopy (CLSM) showed that SH2 domains had a diffused cytoplasmic distribution in RBL-2H3 cells. After antigen stimulation, a markedly different cellular distribution was observed in the cells stained with two IgGs, 12E and 32D.12E IgG stained the plasma membranes, while 32D IgG stained small granules in the cytoplasm. Fluorescence images stained by 12E IgG were similar to those observed by anti-Lyn, not anti-Src. The resulsts suggested that some tyrosine kinases should cluster along the plasma membranes after antigen stimulation. The surface expression of CD63 antigen reflected the degranulation in RBL-2H3 cells. The expression was also observed in P815 mastocytoma cells with normal IgE receptors, but not in P815 variant cells with IgE receptors which were missing a C-terminal cytoplasmic domains of beta and gamma subunit. Secretory structures were observed by atomic force microscopy (AFM). AFM images showed that there were at least two kinds of pits, a small one (0.01mum^2) and a large one (1 mum^2). The former was considered to be the receptor endocytosis and the latter was secretory granules.
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