Amano T, Furuno T, Hirashima N, Ohyama N, Nakanishi M
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
J Biochem. 2001 May;129(5):739-44. doi: 10.1093/oxfordjournals.jbchem.a002914.
CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.
CD63存在于静息嗜碱性粒细胞、肥大细胞和血小板的嗜碱性颗粒膜上，也存在于这些细胞的质膜上。我们构建了一个CD63-绿色荧光蛋白（GFP）质粒，并将其导入大鼠嗜碱性白血病（RBL-2H3）细胞中，以观察脱颗粒过程中CD63的运动。通过共聚焦激光扫描显微镜（CLSM）在活的RBL细胞中研究CD63-GFP的运动。GFP与CD63的C末端偶联的CD63-GFP位于RBL细胞的颗粒膜和质膜上。细胞质中荧光颗粒的直径在0.5至1.5微米之间变化。在抗原刺激之前，大多数带有CD63-GFP的颗粒在RBL细胞中几乎不移动。然而，抗原刺激后，质膜剧烈褶皱，颗粒剧烈移动。它们在几分钟内到达质膜并立即与之融合。对每个颗粒运动的分析为脱颗粒的基本过程提供了新的见解。抗原刺激时颗粒向质膜移动的速度经计算为0.1±0 .02微米/秒。这表明，如果细胞直径为20微米，颗粒能够在2或3分钟内到达质膜。
