Anionic sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization

Silva, P.Pinto Da; Moss, Paul S.; Fudenberg, H. Hugh

doi:10.1016/0014-4827(73)90119-5

Cited by 110 publications

(21 citation statements)

References 37 publications

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“…Although the chemical nature of the structures represented by the membrane particles of myelin cannot be established in the present study, investigations of the chemical nature of the particles in other membranes have always revealed that they are protein-containing structures (21)(22)(23)(24)(25).…”

Section: Resultsmentioning

confidence: 76%

Membrane particles on fracture faces of frozen myelin.

Silva¹,

Miller²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Freeze-fracture electron microscopy reveals constant and widespread presence of membrane particles on the fracture faces of frozen myelin. In unfixed myelin frozen shortly after dissection the distribution of the particles is uniform. In glutaraldehyde-fixed and/or glycerol-impregnated myelin the particles frequently occupy a network interspersed with circumscribed particle-free ateas of variable dimension. In these membranes the network of particles is propagated radially across many lamellae. Particle-rich areas are closely apposed and contrast with frequent delamination of adjacent particle-free regions. We propose that, as in other plasma membranes, the particles of myelin represent protein-containing structures intercalated across the hydrophobic matrix of a membrane with bilayer organization. Our results indicate that these structures contain sites which mutually interact at the surface of apposed membranes and may be important in maintaining the organizational integrity of myelin. Freeze-fracture splits the lipid bilayer regions of the membrane and allows electron microscopic examination of hydrophobic membrane domains and the detection of sites (membrane-intercalated particles) where the bilayer is interrupted (1). The single exception to the presence of particles in plasma membranes was reported by Branton in a study of the fracture faces of frozen myelin (2). These were reported as being totally free of particles, and their featureless appearance made them virtually undistinguishable from the fracture faces observed in lamellar phase, unsaturated phospholipids (3). Interpretation of the x-ray diffraction patterns of myelin supported also the assumption of an uninterrupted bilayer (4) and, although admitting the possibility of small amounts of protein extending across it, explained asymmetries of the electron density profile as the result of an asymmetric lipid distribution (4). These electron microscopic and x-ray diffraction interpretations were consistent with the accepted function of myelin, because they provided a sheath of high electrical resistance which insulated the axon (5, 6). Anatomically, however, electron microscopic examination of myelin from central and/or peripheral nervous systems revealed structural complexities not expected from simple multilayer systems: (a) paranodal myelin/axon and myelin/ myelin specializations (7) MATERIALS AND METHODSRat (Lewis; 12-15 weeks) sciatic and optic nerves were dissected from sodium-pentobarbital-anesthetized animals and fixed immediately in 2% glutaraldehyde (Fisher Scientific Co., Pittsburgh, Pa.) in phosphate-buffered saline at pH 7.3 for 1 hr at 37'. Frog (Rana pipiens) sciatic nerves were dissected from pithed animals and fixed as above. Garfish (Lepisosteus osseus) olfactory and IV nerves were dissected from decapitated fish and fixed in 2% glutaraldehyde in Ringer's solution (12). After fixation the material was gradually impregnated by dropwise addition of 25% glycerol in phosphate-buffered saline or Ringer's solution over a period o...

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Section: Resultsmentioning

confidence: 76%

Membrane particles on fracture faces of frozen myelin.

Silva¹,

Miller²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…However, in these studies membrane skeletons were obtained by Triton extraction of Spectrin-immunogold labelled erythrocytes (unfixed, unglycerinated erythrocyte ghosts and, therefore, did not provide views of the organization of the cytoskeleton associated in situ to the cytoplasmic surface of the erythrocyte membrane. High-resolution, limited views of the inner surface of erythrocyte membrane are provided by freeze-etching of erythrocyte ghosts (13,15,16). Lang et al (8) and Nermut (13) obtained in situ views of the membrane skeleton, after lysis and squirting of erythrocytes attached to positively charged supports.…”

Section: Discussionmentioning

confidence: 99%

Fracture-flip/Triton X-100 reveals the cytoplasmic surface of human erythrocyte membranes.

Fujimoto

Silva

1992

Acta Histochem. Cytochem

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We propose the use of fracture-flip combined with Triton X-100 extraction to visualize the cytoplasmic surface of plasma membranes.Unfixed human erythrocytes were freeze-fractured, carbon-cast, and thawed. The carbon casts, along with attached freeze-fractured erythrocytes, were treated with 2% Triton X-100 to solubilize unfractured plasma membranes and to release haemoglobin. After repeated washing, the carbon-casts, along with attached protoplasmic and exoplasmic membrane halves, were picked on grids, flipped, and Pt-shadowed.Our method leads to extended views of the cytoplasmic surface revealing the fibrilar network that laminates the inner surface of the erythrocyte membrane. Spectrin immunogold labelling of fractured, carbon cast erythrocytes shows that the colloidal gold particles are associated with the fibrilar network at the cytoplasmic surface.Removal of membrane skeletal elements including spectrin by treatment with a low ionic strength buffer containing EDTA leads to loss of the network and reveals globular particles on the cytoplasmic surface of the membrane. These globular particles contained band 3, as shown by immunogold labelling.Our method can be extended to both the ultrastructural observation and the cytochemistry of the cytoplasmic surfaces of other biomembranes.Recently, we have developed 'Fracture-Flip', a method that provides extended, macromolecular resolution views of cell surfaces (1,2,5,14). Fracture-flip proceeds as follows: (1) Freezing: Fixed or unfixed cells are frozen.(2) Freeze-fracture: Freeze-fracture splits the membranes into protoplasmic and exoplasmic halves. (3) Carbon-fixation: The exposed EF faces are evaporated with carbon. This produces a carbon cast with stabilized membrane halves. (4) Washing, flipping (inversion), and Pt-shadowing of the carbon cast: The carbon casts are thawed. Contrary to conventional freeze-fracture, the carbon casts are not digested with acids or bases. Instead, they are repeatedly rinsed in distilled water to wash away unfractured cells. The exoplasmic halves of membranes remain attached to the carbon casts, which are transferred to electron microscope grids. The carbon casts on grids are flipped (inverted). Pt-shadowing creates the informative three-dimensional images of membrane surfaces.As developed, fracture-flip cannot reveal the inner, cytoplasmic surface of the membrane because the cell body remains attached to the inner, protoplasmic membrane half. We report here a method to overcome this obstacle. Briefly, we use unfixed cells that, after freeze-fracture and carbon-stabilization of apolar domains, are treated with Triton X-100. We reasoned that while the detergent would dissolve unfixed, unfractured membranes, it would not reach, micellize, and extract split membranes, as their apolar domains were now positioned against, and stabilized by, their carbon casts. After washing, carbon casts with their stabilized membrane halves are imaged by Pt/C evaporation. Our results provide views of the membrane skeleton of erythrocytes in situ. Immun...

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“…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)(7)(8)(9)(10)(11)(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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Silva¹,

Parkison²,

Dwyer³

1981

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Anionic sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization

Cited by 110 publications

References 37 publications

Membrane particles on fracture faces of frozen myelin.

Membrane particles on fracture faces of frozen myelin.

Fracture-flip/Triton X-100 reveals the cytoplasmic surface of human erythrocyte membranes.

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

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