Freeze-cleave(Freeze-etch)法における割面のcomplementarityについて.Preliminary impression replicaによる研究.

Abstract

Evidences have been accumulated that cell membranes in a frozen state are split and its interior structures are revealed. It has been proved by double replica method th at there is a unique cleavage along membranes and that convex faces, which are referred as the A face, and concave faces, the B face, are reciprocal ones of fractured membranes. Elevations and depressions revealed on one face by fracture through a frozen tissue are matched to those on the other, but there are little pits on the exposed membrane surfac es complementary to particles on the reciprocal ones, except for the junctional are as and certain plasma membranes. It might be due to difficulties inherent in this technique to reveal small and shallow indentations. Preliminary impression replica, relief of which i s perfectly matched to that of the fractured face, may be of value in studying origin of imperfect matching of freeze-cleave images on the reciprocal fracture faces.Rat brain was perfused through the heart with 25 per cent glycerol i n saline solution and small blocks were dissected from the cerebral cortex. Fractured face of a frozen tissue block was replicated without shadowing using the modified freeze-etch appa ratus of Bullivant and Ames type. The replica was backed with silver and collodion film to enforce and distinguish the back of the replica. After dissolvement of tissue the replica was shadowed on the side of tissue, and collodion film and silver were succes sively removed by amyl acetate and nitric acid respectively.On the preliminary impression replica, relief reverse to the usual fracture faces is shown and its resolution is nearly as good as that of ordinary ones. Tissue structures shown on the preliminary impression replica are not different from those see n on the freeze-cleave replica, except for surface characteristics.On the freeze-cleave replica the surfac e of the fractured cytoplasm is granular and coarse on one hand, it appears smooth and fine on the preliminary impression replica on the other hand. There are particles on freeze-cleaved membranes and on the preliminary impression replica those are shown as pits, and very few particles are present on membranes, indicating that there are very few matched depressions on fractured membrane faces, for small depressions, if present on the fractured face, are clearly demonstrable by small protrusions on the preliminary impression replicas.From the present experiments, it is shown that relief per fectly matching with fracture face is different from that seen on the freeze-cleave replica when relation of particles to depressions is taken into account. As the reciprocal freeze-cleave replicas prepared by double replica method, appear quite alike and indistinguishable from each other, it is obvious that reciprocal fracture faces are no longer perfectly matching though these are generated by single fracture. Since elements which lose complementarity on the fracture face are of the protein nature, such as particles on the membrane face and microtubules, probably intracytoplasmic proteins also, the loss of complementarity may be explained by plastic deformation of proteins at the time of fracture.