Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

Abstract

A method-"fracture label"-is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the. inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragd from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new. anionic-sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed. by freeze-fracture of isolated cells and tissues.Freeze-fracture splits membranes along their hydrophobic interior, following the juncture ofend groups provided by a membrane continuum with bilayer organization (1-4). Although fracture of the bilayer produces smooth faces, in all biological membranes these faces are interrupted by particulate components ("membrane intercalated particles" or "intramembranous particles") and, in most, by subtler rugosities ["subparticles" (5)]. Combined use ofimmunochemical and cytochemical techniques with freeze-etching methods, and freeze-fracture observation of reconstituted membrane preparations, demonstrate that, in the systems used, the particles represent the sites ofintegral transmembrane proteins (6-12). Because of the qualitative similarity offreeze-fracture images produced by all biological membranes, the particles are assumed to represent local structural asymmetries in the fracture process provoked by integral membrane.proteins and, possibly, their-associated lipids (3, 4). In the erythrocyte membrane the particles represent the site of the two main integral transmembrane proteins (glycophorin and band III component) which bear AB(H) antigens, influenza virus receptors, wheat germ agglutinin and concanavalin A (Con A) binding sites, as well as anionic sites (8)(9)(10)(11)(13)(14)(15).Although splitting of the bilayer continuum by freeze-fracture (and, in consequence, the partition of peripheral membrane proteins) is established, less is known about the fracture behavior of integral membrane proteins (3, 16)-in particular, their antigens and lectin binding sites at the surface (17). Most particles are generally associated with the inner membrane half (1, 2)-i. e., they are observed on the protoplasmic or P face (18). This may be the result ofa stochastic process or reflect individual differences of fracture behavior of the components in each particle or both. On face P the particles represent, at least in part, portions of integral proteins located at the outer (exoplasmic, E) membrane half. It is unknown whether the particles contain hydrophilic surface groups ofintegral proteins dragged, during fracture, from the outer surface. We have labeled freezefractured erythrocytes and repor...

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

“…The analysis of the thin-section morphology of membrane fracture faces was the object of previous publications (15,16). The present results show that, as with other membranes, in pancreatic cells thin sections of fracture faces appear as interrupted unit membrane profiles.…”

“…2). As in other membranes (15,16), thin sections of protoplasmic faces consist of a series of short segments with an interrupted unit membrane (trilaminar) profile (Figs . 5 and 13).…”

“…Upon fracture, each face is free from the peripheral membrane components associated with the other membrane half. In addition, chemical groups at the outer surface that are part of integral, membrane-traversing proteins can be labeled on the protoplasmic face as they can be dragged during the fracture process across the exoplasmic half of the membrane and labeled on the protoplasmic face (15). Partition of exoplasmic surface sites associated with integral membranetraversing proteins appears, however, to depend on local conditions such as the effectiveness of anchoring to components at the cytoplasmic surface : for instance, in human erythrocytes Con A binding sites (associated with Band III component) are preferentially labeled on the protoplasmic face (15) and WGA (associated with glycophorin) on the exoplasmic face (Pinto da Silva and Torrisi, manuscript in preparation) .…”

“…Recently, we showed that cytochemical and immunochemical techniques can be combined with freeze-fracture to gain generalized access for direct labeling of the fracture faces of biological membranes as well as of exposed groups in crossfractured cytoplasm (14)(15)(16) . The results of these "fracturelabel" techniques can be assessed either by observation of thin sections of freeze-fractured specimens ("thin-section fracturelabel") (15,16) or of platinum/carbon replicas of critical point dried, freeze-fractured preparations ("critical-point-drying fracture-label") (14).…”

“…The results of these "fracturelabel" techniques can be assessed either by observation of thin sections of freeze-fractured specimens ("thin-section fracturelabel") (15,16) or of platinum/carbon replicas of critical point dried, freeze-fractured preparations ("critical-point-drying fracture-label") (14).…”