Changes in the plasma membrane complex following the injury of single cells of Amoeba proteus were examined with the electron microscope . Two types of injury were employed in this study ; cells were either pinched ("cut") in half or speared with a glass microneedle, and quickly fixed . Speared cells, when fixed in the presence of the ruthenium violet (a derivative of ruthenium red), revealed the presence of an extra trilaminar structure outside of each cell . This structure, called the "new membrane," was separated from the plasma membrane complex by a distance of less than a micron. The trilaminar structure of the new membrane strikingly resembled the image of the plasma membrane in all cells examined, except for its increased width (30%) . This new membrane appeared nearly to surround the injured amebae. Attempts were made to demonstrate the possible origin of the new membrane, its reality, and its sensitivity to calcium . Also, some evidence is shown concerning the role of the small dense droplets (100-1200 A in diameter) normally present in the cytoplasm of amebae . Their frequent contact with the plasma membrane of the cell as the result of injury is interpreted as indicating their involvement in the formation and expansion of the plasma membrane .
Ruthenium violet, closely related to ruthenium red, supplements the ultrastructural knowledge of the plasnia membrane complex. Amoebae throughout were handled individually with braking pipettes and were exposed to ruthenium violet alive, during fixation with acrolein and 0~0 4 , or during dehydration. Ruthenium violet was less toxic than ruthenium red but still killed the amoebae. Conventional methods reveal a filamentous layer 2000 A thick, an amorphous layer 150 A thick, and a typical trilaminar plasma membrane (48 A center-to-center). Ruthenium violet binds to the plasma membrane, and to the extraneous coats revealing globules in the filamentous layer. The diameter of the globules decreased according to the stage of processing at which the amoebae first encountered ruthenium violet; they were 1200 A in diameter when amoebae were alive, 600 A in acrolein and 300 A in dehydration. The appearance of the filamentous layer varied when ruthenium violet was replaced by very pure ruthenium red or red containing ruthenium brown (typical of commercial ruthenium red). The globules could be demonstrated without using ruthenium dyes when amoebae were treated after fixation with uranyl acetate or phosphotungstic acid. The relationship of extraneous coats of amoebae is compared with the coats and laminae of animal tissue cells.Amoebae have been objects of scientific interest and study for years, and many fascinating features of their biology, such as pinocytosis, movement, and nuclearcytoplasmic interactions have been carefully studied (Whitelock and Hirshfield, '59). It is known that the plasma membrane complex of the amoebae contains polysaccharides as indicated by a positive periodic acid-Schiff reaction (PAS) (Pappas, '54), that it contains a sulfated polysaccharide incorporating mannose (Marshall and Nachmias, ' 6 5 ) , and the ultrastructure has been carefully described (Pappas, '59; Mercer, '59; Brandt and Pappas, '62; Nachmias, '68).The large, free-living amoebae A. proteus and Chaos chaos were chosen as convenient organisms with which to investigate certain aspects of the work on ruthenium red (RR) and ruthenium violet (RV) which were inconvenient with animal tissues, and at the same time to contribute further understanding to the ultrastructure oE amoebae. Amoebae were particularly useful with ruthenium violet (RV) since this substance is precipitated from solution by the chloride content of the extracellular fluids in animal tissues (Luft, '71, I); amoebae, however, tolerate even distilled water well and solutions of RV in distilled water are stable. Also, the use of freeliving, single-celled organisms avoided the complication of unpredictable diffusion of ruthenium red (RR) into the intercellular spaces of animal tissues (Luft, '71, 11), thus permitting separate evaluation of the factors of time and concentration in exposure of cells to RR and RV. Finally, use of amoebae permitted testing for toxicity of both RR and RV as well as for their effects on pinocytosis. 418BARBARA SZUBINSKA AND JOHN H. LUFT ...
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