Confocal and Probe Microscopy for Studying Immune Responses

SH2结构域存在于细胞质酪氨酸激酶中，也存在于参与信号转导的各种蛋白质中。我们在此制备了针对SH2结构域高度保守序列（GTFLVRESETTK）的单克隆抗体。共聚焦激光显微镜（CLSM）的荧光图像显示，RBL-2H3细胞中SH2结构域呈弥漫性分布。抗原刺激后，12E和32D两种igg染色细胞的细胞分布明显不同。12E IgG染色质膜，32D IgG染色细胞质内小颗粒。12E IgG染色的荧光图像与anti-Lyn相似，而与anti-Src不同。结果提示抗原刺激后，部分酪氨酸激酶沿质膜聚集。CD63抗原的表面表达反映了RBL-2H3细胞的脱粒。在具有正常IgE受体的P815肥大细胞瘤细胞中也观察到表达，而在具有缺失β和γ亚基c端胞质结构域的IgE受体的P815变异细胞中未观察到表达。用原子力显微镜（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，后者是分泌颗粒作用。

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.