Electron Microscopic Stains as Probes of the Surface Charge of Mitochondrial Outer Membrane Channels

Abstract

To enhance contrast for electron microscopic imaging, biological structures are often negatively stained, i.e., dried down in a thin layer of a glass-forming heavy-metal salt. An ideal negative stain is one which fills in the surface relief of specimen molecules. However, electron-microscopic stain distribution may also reflect positive staining, e.g., binding of charged stain molecules to oppositely charged sites on the specimen. Recent model studies on proteins of known primary structure (1-4) have shown that both kinds of staining may occur simultaneously in crystalline and paracrystalline arrays. We have found (5) that outer membranes isolated from Neurospora mitochondria contain distinctive ordered arrays of roughly circular features, 2-3 nm in diam (Fig. 1), which accumulate a wide variety of electron microscopic stains. The nonspecific accumulation of stain suggests that these features are topological, i.e., depressions in the membrane surface that might represent the openings of hydrophilic pores. Their pore nature is supported by recent immunological evidence (6) that the arrays are composed of the same 31,000-dalton polypeptide which, when extracted from the outer mitochondrial membranes, forms ion channels in phospholipid bilayers. Unlike the nonspecific accumulation of stain inside the putative channels, there are indications of specificity in the distribution of different stains elsewhere on the membrane arrays (5). The existence of specific distribution patterns for different stains has been confirmed by multivariate statistical analysis of the computed diffraction patterns from many electron images of differently stained outer mitochondrial membrane arrays (7). The results of the present report demonstrate that the distribution patterns of two of these stains, potassium phosphotungstate (KPT) and uranyl acetate (UA), may be explained in terms of the local surface charge at different sites on the channel arrays.