Effects of filipin, digitonin, and polymyxin b on plasma membrane of ram spermatozoa--an EM study.

The effects of three membrane-active agents, filipin, digitonin, and polymyxin B on the plasma membrane of ram spermatozoa have been studied by freeze-etch electron microscopy. Filipin and digitonin both reacted with cholesterol and caused visible membrane modification in cholesterol-rich regions, with filipin being a more specific agent than digitonin. Polymyxin B, which is known to interact specifically with negatively charged phospholipids of bacterial membranes, exhibited a selective binding and subsequent modification of sperm plasma membranes. This binding was shown to be inhibited in the presence of 1 mmol/1 Ca2+. We hence propose that both filipin and polymyxin B are useful cytological markers for specific biological molecules in eukaryote membranes--filipin for cholesterol, and polymyxin B for negatively charged phospholipids.

Fine structure of the gap junction in the tunicate heart.

The plasma membranes of the tunicate heart exhibit an abundance of macular gap junctions distributed widely over the membrane surface. A study of these junctions by the freeze-etch technique was undertaken in an effort to elucidate the fine structure of this important membrane modification in a primitive heart. In cross or near-cross fractured junctions the junctional particles in contiguous membranes appear to be paired in register and to meet in the midline. In favorable face views, the junctional particles are seen to be disposed in hexagonal array. The individual particles display a distinct rosette-like substructure consistent with a six-membered ring of globular protein molecules clustered around a central channel. Similar junctional-type particles can be found in nonjunctional areas of membrane suggesting that the transport mechanism which they may represent is not restricted to the gap junction.

Mesothelial intercellular junctions and pathways.

Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the "stomata" concept observed by early German and contemporary anatomists. "Occludens" junctions of the leaky type are seen though their macular or zonular nature is yet to be established.

Freeze-fracturing and freeze-etching of cardiac myosin filaments.

Myofilament structure was studied in freeze-etch replicas of unfixed, glycerinated beef cardiac muscle. The information which is revealed depends upon the direction of metal shadowing in relation to the filament axis. Shadows oblique to this axis reveal that the outer surface of a longitudinal half of a thick filament comprises three, sometimes four, rows of myosin molecules. These molecules are generally assembled in a braided manner with both left and right-handed helical components. Occasionally a more parallel to the myofilament axis reveal cross-bridges linking thick and thin filaments. These bridges are readily detectable by optical diffraction techniques, giving an axial bridge spacing of approximately 40 nm. In unetched preparations cross bridges appear as vertical rows of beads. In all replicas the effects of plastic deformation of proteins must be considered.