Kim Hyonchol, Arakawa Hideo, Hatae Noriyuki, Sugimoto Yukihiko, Matsumoto Osamu, Osada Toshiya, Ichikawa Atsushi, Ikai Atsushi
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama 226-8501, Japan.
Ultramicroscopy. 2006 Jun-Jul;106(8-9):652-62. doi: 10.1016/j.ultramic.2005.12.007. Epub 2006 Apr 18.
The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.
采用原子力显微镜（AFM）对活细胞表面EP3受体的分布进行了定量研究。将绿色荧光蛋白（GFP）导入CHO细胞上EP3受体的细胞外区域。使用微珠作为探针以确保一定的接触面积，微珠表面包被有抗GFP抗体。记录抗体与细胞表面GFP分子之间的相互作用以观察受体的分布。结果表明，EP3受体在CHO细胞表面并非均匀分布，而是呈小斑块状分布，这与免疫组织化学观察结果一致。对细胞表面同一区域的重复测量证实，在实验过程中受体不太可能从细胞膜上被提取出来。利用无压缩力谱成功地在细胞表面测量了GFP与抗GFP抗体之间的单分子相互作用。估计破坏单个分子对所需的分离功值约为1.5×10（-18）J。假设细胞表面积约为5000平方微米，利用该值估计CHO细胞表面EP3受体的数量约为1×10（4）。这些结果表明，通过AFM进行力测量可以对活细胞表面的受体数量进行定量。
