DIRECT MEASUREMENT OF DIMENSIONS OF CELL ADHESION MOLECULES ON CELL SURFACE

(部分由美国国立卫生研究院R01 GM 23484向卡罗尔·威尔斯提供支持；NIH R01 GM 49530至加里·邓尼；明尼苏达大学研究生院，以及 明尼苏达医学基金会在日立S-900型飞机上安装立体管 对S.Erlandson进行定量测量的FESEM)， 高分辨率场发射扫描电子显微镜免疫金标记法研究 已经展示了几个不同的空间分布 细胞表面的细胞黏附分子(CAM)，但不幸的是 分子本身的分子拓扑结构并不是 保存下来的，只有用于鉴定的胶体金标记。 设计了一种使用分散的人血小板的模型系统 包括整合素P-选择素在内的特殊形状CAM的检测 GPIIb-IIIa和GPI-IX复合体。血小板的前固定是 然后是间接免疫金标记法，然后是骤降冷冻 冷冻固定术。血小板膜表面用电子显微镜观察 水升华后的双层包覆法 Walther(在2-3 nm的金属和5 nm的碳的阴影下)， 然后用日立S-900型背散射电子成像仪进行检查 场发射低温扫描电子显微镜。产生热离子金属阴影 细胞外表面良好的地形对比度 血小板。利用立体成像，P-选择素分子被可视化 当杆状分子突出在膜表面上方时， 整合素，GPIIb-IIIa，被认为是上面突出的小突起 膜，而GP1ba被视为更大的线性表面的一部分 很复杂。我们使用的是Sterecon系统和低温扫描电子显微镜 图像来直接测量分子形状的长度。 Erlandsen博士在BMIRR参观了两天。表面蛋白是 在四个实验中测量，大小与生化一致 数据。Erlandsen博士在他自己的实验室里有Sterecon，我们正在扩展 它的表面积测量能力更好地适应了这一点 项目(见综合安全分遣队分项目“立体声传播”)。

(Supported in part by NIH R01 GM 23484 to Carol Wells; NIH R01 GM 49530 to Gary Dunny; University of Minnesota Graduate School, and Minnesota Medical Foundation Sterecon Installation on Hitachi S-900 FESEM for Quantitative Measurement to S. Erlandson) In previous work, immunogold labeling studies with high-resolution field-emission SEM have demonstrated the spatial distribution of several different cell-adhesion molecules (CAM) on the cell surface, but unfortunately the molecular topography of the molecules themselves was not preserved, only the colloidal gold markers used for identification. A model system using spread human platelets was devised for the detection of distinctly-shaped CAM including P-selectin, the integrin GPIIb-IIIa, and the GPI-IX complex. Prefixation of platelets was followed by indirect immunogold labeling, then by plunge freezing for cryoimmobilization. The membrane surface of platelets was revealed by sublimation of water followed by the double-layer coating method of Walther (shadowing with 2-3 nm of metal followed by 5 nm of carbon), then examination by backscatter electron imaging using a Hitachi S-900 field emission cryo-SEM. Thermionic metal shadowing produced excellent topographical contrast of the extracellular surface of platelets. Using stereo imaging, P-selectin molecules were visualized as rod-shaped molecules projecting above the membrane surface, the integrin, GPIIb-IIIa, was seen as small protuberances projecting above the membrane, and GP1ba was seen as part of a larger linear surface complex. We used the Sterecon system with ste reopairs of cryo-SEM images to make direct measurements of the length of molecular shapes. Dr. Erlandsen visited the BMIRR for two days. Surface proteins were measured in four experiments, and the sizes agreed with biochemical data. Dr. Erlandsen has Sterecon in his own lab, and we are extending its capabilities for surface area measurements to better suit this project (see DIS subproject "Sterecon Dissemination").