Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Silva, P P da; Torrisi, Maria Rosaria

doi:10.1083/jcb.93.2.463

Cited by 37 publications

(29 citation statements)

References 18 publications

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“…They contrast with the images seen in mens, reorganization of freeze-fracture membrane halves re-suits in a rugose structure (Fig. 9) where membrane-intercalated particles are not identifiable (27,28,(41)(42)(43) . Observation of replicas of boar sperm flagella illustrated clearly the high density of both Con A and WGA receptors along protoplasmic membrane halves of the different segments of the sperm tail (Figs .…”

Section: Thin-section Fracture-labelcontrasting

confidence: 41%

“…As freeze-fracture splits biological membranes through its hydrophobic plane (46,47), we conclude that labeling on protoplasmic halves results from dragging, during fracture, of oligosaccharides of transmembrane glycoproteins across the outer (exoplasmic) half of the membrane. Previous comparisons on the partition of the major integral proteins of human erythrocytes with freezefracture membrane halves suggested the operation of a stochastic process that reflects the relative expression of each species of transmembrane protein at each side of the membrane as well as its interaction with components of the membrane skeleton (26,42) . We showed that glycophorin, preferentially expressed at the outer surface and without linkages to the membrane skeleton (48,49), partitions preferentially with the outer half of the erythrocyte membrane while band 3, which has a large hydrophilic segment exposed at the inner surface and known associations to the membrane skeleton (50,51), partitions preferentially with the inner half of the membrane (26,42).…”

Section: Discussionmentioning

confidence: 99%

“…Previous comparisons on the partition of the major integral proteins of human erythrocytes with freezefracture membrane halves suggested the operation of a stochastic process that reflects the relative expression of each species of transmembrane protein at each side of the membrane as well as its interaction with components of the membrane skeleton (26,42) . We showed that glycophorin, preferentially expressed at the outer surface and without linkages to the membrane skeleton (48,49), partitions preferentially with the outer half of the erythrocyte membrane while band 3, which has a large hydrophilic segment exposed at the inner surface and known associations to the membrane skeleton (50,51), partitions preferentially with the inner half of the membrane (26,42). Should this interpretation prove correct, lectin labeling of protoplasmic membrane halves suggests that the transmembrane glycoproteins of the sperm flagella are preferentially expressed at the inner surface and/or linked to submembranous components.…”

Section: Discussionmentioning

confidence: 99%

“…The pellet of BSA cells was gelled by the addition of glutaraldehyde to a final concentration of 1 %. In some samples, glutaraldehyde-fixed human erythrocytes were mixed with the sperm cells as an internal control ofthe freeze-fracture process: patterns of lectin labeling of freeze-fractured erythrocytes were previously established in this laboratory (42) ; erythrocytes are a reliable marker for surfaces exposed in gels by slicing at room temperature because, in these instances, the red blood cells are always separated in toto (never cross-fractured) with gel surfaces . The gels were sliced, impregnated in 30% glycerol in PBS (3 h), frozen in partially solidified Freon 22, and fractured in liquid nitrogen with a glass pestle (27,29,33).…”

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confidence: 99%

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High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

Águas¹,

Silva²

1984

The Journal of Cell Biology

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Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin ; the lectins were visualized with protein-gold complexes . Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane . A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer . Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton .Flagella of eucaryotic cells are specialized extensions of the cell body composed of similar basic components: a central axoneme, a thin layer of cytoplasm that may include mitochondria, accessory fibers or paraflagellar rods, and the limiting plasma membrane (1-4). They are responsible for two independent forms of whole-cell movement : swimming propelled by coordinated bending ofthe flagellum (4) and gliding on the surface of a solid substrate (5-7). Swimming is the result of sliding of axonemal microtubules, as characterized in a number of elegant studies (8-12) . Gliding was first described in Chlamydomonas by Lewin (5) and appears to depend on the dynamics ofthe flagellar surface in association with cytoskeleton structures (7) .Flagellar surface motility is expressed by the saltatory movement of exogenous markers (polysterene microspheres or bacteria) (13). Bloodgood proposed that the markers bind to putative "motility-coupled cell-surface receptors" (7, 13) . The density of these receptors slowly decreases upon incubation of flagella in inhibitors of protein synthesis (cycloheximide) and protein glycosylation (tunicamycin) (6,14) . Pronase digestion of a high-molecular-weight glycoprotein of the flagellar surface causes reversible loss of gliding ability (6, 15) . Structural studies on flagella reported the presence of submembranous cytoskeleton-like elements that, in some cells, may connect the plasma membrane with the outer doublet microtubules of the axoneme (1)(2)(3)(4)(16)(17)(18) . ATPase activity was also found on the submembranous cytoplasm of flagella (19)(20)(21)(22)(23)(24)(25) .Current models on the coupling of surface receptors with the cytoskeleton postulate the existence of transmembrane THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 655-660 p The Rockefeller University Press 0021-9525/84/08/0655/06 $1 .00 glycoproteins on the flagellar membrane (3,14) . This topology of glycoproteins in the membrane would allow the same molecule to express (on its outer portion) the surface receptors and to be accessible (on its cytoplasmic portion) for interaction with the membrane skeleton or with axonemal microtubules. Here, we search for the topology of glycoconjugates on the flagellar s...

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Section: Thin-section Fracture-labelcontrasting

confidence: 41%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

Águas¹,

Silva²

1984

The Journal of Cell Biology

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“…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 along the plane of the membrane (19)(20)(21)(22)(23). Because labeling is done after freeze-fracture, fracture-label was not intended as a substitute for surface labeling.…”

mentioning

confidence: 99%

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

Silva¹,

Kan²

1984

The Journal of Cell Biology

125

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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 ...

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Wheat germ agglutinin fracture‐label of lymphocyte plasma and intracellular membranes

Torrisi¹,

Pavan²,

Lotti³

et al. 1986

J. Elec. Microsc. Tech.

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Conventional lectin cytochemistry or immunocytochemistry can be associated with freeze-fracture in fracture-label techniques: The thin section fracture-label and the critical point drying fracture-label. In the first method, which is particularly useful for investigating intracellular membranes, cells or tissues are freeze-fractured, thawed, labeled, and embedded in resin. Wheat germ agglutinin labeling on freeze-fractured human lymphocytes confirms the existence of two compartments of intracellular membranes, based on the sialoglycocomponent content.

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Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Cited by 37 publications

References 18 publications

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

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

Wheat germ agglutinin fracture‐label of lymphocyte plasma and intracellular membranes

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