The cytoskeleton of digitonin-treated rat hepatocytes.

Abstract

Treatment of isolated rat hepatocytes with low concentrations of digitonin increases the permeability of the plasma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentrations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes. Digitonin, a steroid glycoside, increases the permeability of different types of cells to inorganic ions (1, 2, *), metabolites (1-4, *), and enzymes (3,5,6). This effect is believed to be the result of the binding of digitonin to cholesterol and other 3-hydroxysterols present in the plasma membrane (7). Because the molar ratio of cholesterol to phospholipid in eukaryotic plasma membranes is severalfold or manyfold greater than in intracellular membranes (8), treatment of intact cells with low concentrations of digitonin can be expected to bring about a selective increase in the permeability of the plasma membrane. This property has been used in this and other laboratories to make measurements of transport activities by mitochondria (1, 2, 9, *) and endoplasmic reticulum (9) in cells in situ. Digitonin offers a major advantage over other commonly used agents, such as Triton, glycerol, or toluene, because the latter are relatively nonspecific in their effects on different types of cell membranes.Experiments described in this paper demonstrate another useful consequence of the treatment of cells with digitoninnamely, greatly enhanced electron microscopic visualization of certain intracellular structures, particularly the cytoskeletal network of filaments, which are normally difficult to observe due to the presence of dark-staining cytosolic protein. Such pretreatment of hepatocytes with low levels of digitonin causes no loss or impairment of the normal morphology of membrane-limited organelles, such as mitochondria, endoplasmic reticulum, and lysosomes. The enhanced visualization of intracellular structures as described here reveals some hitherto unreported associations of microfilaments and intermediate filaments within isolated normal hepatocytes, supplementing earlier reports on the role of microfilaments in maintenance of cell shape (10), in endocytosis (11), and in secretion of lipoproteins (10). MATERIALS AND METHODSIsolation...

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