Composite replicas: methodologies for direct evaluation of the relationship between intramembrane and extramembrane structures.

Electron microscopic studies of membrane structure have been facilitated by the recent development of the composite replica technique in which the membrane is freeze-fractured, then inverted and the surface deep-etched and replicated. Examination in stereo of this composite preparation of two replicas with interposed half-membrane and associated surface elements reveals the physical relationship between structures on the surface and within the membrane. Composite replicas of the toad urinary bladder surface demonstrated connections of filamentous glycocalyx elements to intramembrane particles (IMPs). Using a bidirectional shadowing technique, many membrane surface particles also are shown to be associated with underlying IMPs, suggesting that these membrane surface particles are projections of the IMPs above the surface of the membrane. There is evidence that elements whose attachment sites relate to the half-membrane fractured away can be displaced from the membrane surface and lost. Labelling studies using colloidal gold-labelled antibodies were carried out to assess loss of surface mesh from fractured membrane. Gold distributions and amounts were similar in labelled surface replicas, label-fracture specimens, and labelled composite replicas, yet the amount of mesh detected in the composite replicas was less than in the surface replicas. This suggests that while some unlabelled or lightly labelled surface elements can be lost from fractured membranes, ligands stabilize elements and reduce their loss apparently by cross-linking them.