Freeze-fracture cytochemistry in cell biology.
The term freeze-fracture cytochemistry embraces a series of techniques which share the goal of chemical identification of the structural components viewed in freeze-fracture replicas. As one of the major features of freeze fracture is its ability to provide planar views of membranes, a major emphasis in freeze-fracture cytochemistry is to identify integral membrane proteins, study their spatial organization in the membrane plane, and examine their role in dynamic cellular processes. Effective techniques in freeze-fracture cytochemistry, of wide application in cell biology, are now available. These include fracture-label, label fracture, and the freeze-fracture replica immunolabeling technique (FRIL). In fracture-label, samples are frozen and fractured, thawed for labeling, and finally processed for viewing either by critical-point drying and platinum-carbon replication or by thin-section electron microscopy. Label-fracture involves immunogold labeling a cell suspension, processing as for standard freeze-fracture replication, and then examining the replica without removal of the cellular components. Of greatest versatility, however, is the FRIL technique, in which samples are frozen, fractured, and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate (SDS) to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits the distribution of identified components to be viewed superimposed upon high resolution planar views of replicated membrane structure, for both the plasma membrane and intracellular membranes in cells and tissues. Examples of how these techniques have contributed to our understanding of cardiovascular cell function in health and disease are discussed.
Severs
Nicholas J
NJ
Imperial College London, National Heart and Lung Division, London, United Kingdom.
Robenek
Horst
H
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002468:Cell Physiological Phenomena; D005614:Freeze Fracturing / Q000379:methods*; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008954:Models, Biological
D008055:Lipids