Confocal and Probe Microscopy for Studying Immune Responses

用于研究免疫反应的共焦和探针显微镜

基本信息

批准号：
07457531
负责人：
NAKANISHI Mamoru
金额：
$ 4.22万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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.

SH2 结构域存在于细胞质酪氨酸激酶以及参与信号转导的各种蛋白质中。我们在此针对 SH2 结构域中高度保守的序列 (GTFLVRESETTK) 制备了单克隆抗体。共聚焦激光显微镜（CLSM）观察到的荧光图像显示，SH2结构域在RBL-2H3细胞中具有弥散的细胞质分布。抗原刺激后，在用两种 IgG 12E 和 32D 染色的细胞中观察到明显不同的细胞分布。12E IgG 对质膜染色，而 32D IgG 对细胞质中的小颗粒染色。 12E IgG 染色的荧光图像与抗 Lyn 染色的荧光图像相似，而不是抗 Src 染色的荧光图像。结果表明，在抗原刺激后，一些酪氨酸激酶应该沿着质膜聚集。 CD63抗原的表面表达反映了RBL-2H3细胞的脱颗粒情况。在具有正常 IgE 受体的 P815 肥大细胞瘤细胞中也观察到了该表达，但在具有 IgE 受体（缺少 β 和 γ 亚基 C 末端胞质结构域）的 P815 变异细胞中没有观察到该表达。通过原子力显微镜（AFM）观察分泌结构。 AFM 图像显示至少有两种凹坑，一种是小凹坑 (0.01mum^2)，一种是大凹坑 (1 mum^2)。前者被认为是受体内吞作用，后者是分泌颗粒。