DIRECT MEASUREMENT OF DIMENSIONS OF CELL ADHESION MOLECULES ON CELL SURFACE

点击翻译按钮获取中文摘要

(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").