Label-fracture: a method for high resolution labeling of cell surfaces.

Abstract

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (celloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the PtIC replica of the exoplasmic fracture face.Initial applications indicate high resolution (~<15 nm) and exceedingly low background. "Labelfracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.The localization of cell-surface receptors, antigens, and other chemical groups associated with lipid or protein molecules of the plasma membrane is a recognized objective of the cell sciences. Ultrastructural cytochemistry of cell surfaces was first observed in thin sections of cells and tissues, a process that required the assemblage of composites obtained from serial-sectioned cells (1). To overcome this unusually difficult procedure, other techniques were developed that allowed direct views of the surface distribution of the label: (a) plasma membranes collapsed at an air/water interface (2, 3); (b) platinum/carbon replication of cells (air-dried, freeze-dried, or critical-point dried) (4); and (c) platinum/carbon replication of frozen cells after freeze-fracture and sublimation (freeze-etching) (5-9). All these approaches suffered from severe limitations. Collection of unfixed (or lightly fixed) membranes at the air/water interface could not prevent reorganization of receptors (10). Conventional Pt/C replication of dried specimens could only be applied to cell monolayers (or to cells previously attached to a substrate), and drying procedures could cause deformation and collapse of structures (11, 12). Freeze-etching related the surface distribution of receptors and antigens to that of the intramembrane particles revealed by freeze-fracture but could only be performed in systems (generally isolated membranes) capable of withstanding freezing in the absence of cryoprotectants.Over the past few years, we have developed new labeling techniques--fracture-label (13-16)--that allow direct, in situ labeling of freeze-fractured plasma and intracellular membranes. As freeze-fracture splits biomembranes along their bilayered continuum (17, 18), fracture-label techniques can be used to determine the sidedness of membrane components and, in some instances, to identify transmembrane proteins as well as their regionalization ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

“…The required high resolution to visualize the constituents of protein complexes is achieved by transmission electron microscopy (TEM). However, conventional TEM of whole cells is limited by the requirement of thin samples/sections slicing through the plasma membrane 27 , or plasma membrane ripping or breaking off 28,29 resulting in randomly formed small pieces of membrane. The plasma membrane is thus not imaged as a whole, leading to a lack of possibly crucial cellular context information.…”

Herpes simplex virus enhances chemokine function through modulation of receptor trafficking and oligomerization

“…To assess whether SgG2-mediated increase in cell responsiveness to the chemokine was related to changes in the oligomerization state of CXCR4, as well as to address the effect on the receptor by means of a higher resolution technique, we prepared surface replicas by label-fracture of Jurkat T cells 27,28 , which were analysed by electron microscopy (Fig. 7).…”

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

“…They were freeze-fractured at -110°C in a Balzers BAF401 apparatus (Balzers High Vacuum; Balzers, Liechtenstein). The platinumkarbon replicas were observed after extensive rinses in water without digestion (Pinto da Silva and Kan, 1984). For quantitation of caveolae containing crosslinked molecules, the percentage of round bumps containing more than one gold particle within their perimeter was measured manually on enlarged positive prints of randomly selected areas.…”

“…To examine the two-dimensional relationship between the gold particles and caveolae, freeze-fracture replicas were observed without digestion ("label-fracture") (Pinto da Silva and Kan, 1984). In this preparation, immunogold particles binding to the exoplasmic leaflet of the apical plasma membrane are retained and their twodimensional distribution can be observed.…”