Pilot study on construction of the freeze-etching apparatus to create atomic

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This project aimed to make a high quality freeze-etching replica machine and its application to the structural analysis of the cell membrane undercoat. The apparatus developed has ability to control the temperature from 30 to -160 ℃ under extremely high vacuum (1 X 10^<-6> Pa). The microtom mechanism is also organized in this apparatus, which make possible to section in order of micron. This apparatus has been utilized practically for structural analysis of membrane undercoat of culture cells since middle of 2002. The cytoplasmic surface of cell membrane comsisted of various domains such as crathrin coat, caveolae and smooth surfaced depression that were associated with filamentous structures. Actin and spectrin were major constituents of meshwork of membrane undercoat. We succeeded identification of ankyrin, adducin, gelsolin and Arp proteins by using immuno-gold labeling method. Those actin binding proteins were located at the actin filaments, in particular arp proteins were foumd on the branching points. We also succeeded to create 3D reconstruction or animation of membrane undercoat by 90 tilting electron microscopic images taken at 1° interval between +45° and -45°.