Abstract
Visualization of the components of the red cell membranes, and especially the structure of cytoskeletal proteins in situ, has become a requisite in studies of red cell membrane disorders. There has been a search for a consistent and dependable method for detecting these structures. In the present study, the surface replica method was used with transmission electron microscopy to examine the cytoskeletal network of the red cell ghosts of a normal control and patients with a β-spectrin mutant (β-spectrin Le Puy). The surface replica method is well-suited to observation of the cytoskeletal network of the membranes in a nearly native in situ condition. Immunogold labelling with anti-membrane protein antibodies is easily applicable to the identification of each component of the cytoskeletal proteins. The findings obtained under normal and pathological conditions using the surface replica method corresponded with those made by the quick-freeze, deep-etching method.
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Bohn W, Röser K, Hohenberg H, Mannweiler K, Traub P (1993) Cytoskeleton architecture of C6 rat glioma cell subclones differing in intermediate filament protein expression. J Struct Biol 111:48–58
Byers TJ, Branton D (1985) Visualization of the protein associations in the erythrocyte membrane skeleton. Proc Natl Acad Sci USA 82:6153–6157
Coleman TR, Fishkind DJ, Mooseker MS, Morrow JS, (1989) Functional diversity among spectrin isoforms. Cell Motil Cytoskeleton 12:225–247
Delaunay J, Dhermy D (1993) Mutations involving the spectrin heterodimer contact site: clinical expression and alterations in specific function. Semin Hematol 30:21–33
Dodge JT, Mitchell C, Hanahan DJ (1963) The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119–130
Elgsaeter A, Branton D (1974) Intramembrane particle aggregation in erythrocyte ghosts. I. The effect of protein removal. J Cell Biol 63:1018–1030
Elgsaeter A, Shotton DM, Branton D (1976) Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Biochim Biophys Acta 426:101–122
Fairbanks G, Steck TL, Wallach DFH (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617
Gahmberg CG, Taurén G, Virtanen I, Wartiovaara J (1978) Distribution of glycophorin on the surface of human erythrocyte membranes and its association with intramembrane particles: an immunochemical and freeze-fracture study of normal and En(a-) erythrocytes. J Supramol Struct 8:337–347
Gallagher PG, Forget BG (1993) Spectrin genes in health and disease. Semin Hematol 30:4–20
Gallagher PG, Tse WT, Costa F, Scarpa A, Boivin P, Delaunay J, Forget BG (1991) A splice site mutation of the β-spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated β-spectrin chain. J Biol Chem 266:15154–15159
Gallagher PG, Tse WT, Mohandas N, Marchesi SL, Forget BG (1992) Spectrin Providence: a defect of erythrocyte beta spectrin (β2019 Ser→Pro) homozygosity for which is associated with fatal hydrops fetalis. Blood 80 [Suppl 1]:145a
Garbarz M, Tse WT, Gallagher PG, Picat C, Lecomte MC, Galibert F, Dhermy D, Forget BG (1991) Spectrin Rouen (β220−218), a novel shortened β-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation. J Clin Invest 88:76–81
Garbarz M, Boulanger L, Pedroni S, Lecomte MC, Gautero H, Galand C, Boivin P, Feldman L, Dhermy D (1992) Spectrin βTandil, a novel shortened β-chain variant associated with hereditary elliptocytosis is due to a deletional frameshift mutation in the β-spectrin gene. Blood 80:1066–1073
Heuser J (1989) Protocol for 3-D visualization of molecules on mica via the quick-freeze, deep-etch technique. J Electron Microsc Tech 13:244–263
Inoue T, Kanzaki A, Yawata A, Tsuji A, Ata K, Okamoto N, Wada H, Higo I, Sugihara T, Yamada O, Yawata Y (1994) Electron microscopic and physicobiochemical studies on disorganization of the cytoskeletal network and integral protein (band 3) in red cells of band 4.2 deficiency with a mutation (codon 142: GCT→ACT). Int J Hematol 59:157–175
Kanzaki A, Rabodonirina M, Yawata Y, Wilmotte R, Wada H, Ata K, Yamada O, Akatsuka J, Iyori H, Horiguchi M, Nakamura H, Mishima T, Morlé L, Delaunay J. (1992) A deletional frameshift mutation of the β-spectrin gene associated with elliptocytosis in spectrin Tokyo (β220/216). Blood 80:2115–2121
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Liu S-C, Derick LH (1992) Molecular anatomy of the red blood cell membrane skeleton: structure-function relationships. Semin Hematol 29:231–243
Liu S-C, Palek J, Prchal J, Castleberry RP (1981) Altered spectrin dimer-dimer association and instability of erythrocyte membrane skeletons in hereditary pyropoikilocytosis. J Clin Invest 68:597–605
Liu S-C, Windisch P, Kim S, Palek J (1984) Oligomeric states of spectrin in normal erythrocyte membranes: biochemical and electron microscopic studies. Cell 37:587–594
Liu S-C, Derick LH, Palek J (1987) Visualization of the hexagonal lattice in the erythrocyte membrane skeleton. J Cell Biol 104:527–536
Liu S-C, Derick LH, Agre P, Palek J (1990) Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis and pyropoikilocytosis. Blood 76:198–205
Maréchal J, Wada H, Koffa T, Kanzaki A, Wilmotte R, Ikoma K, Yawata A, Inoue T, Takanashi K, Miura A, Alloisio N, Delaunay J, Yawata, Y (1994) Hereditary elliptocytosis associated with spectrin Le Puy in a Japanese family. Ultrastructural aspect of the red cell skeleton. Eur J Haematol 52:92–98
Nermut MV (1981) Visualization of the “membrane skeleton” in human erythrocytes by freeze-etching. Eur J Cell Biol 25:265–271
Ohno S, Terada N, Fujii Y, Ueda H, Kuramoto H, Kamisawa N (1993) Immunocytochemical study of membrane skeletons in abnormally shaped erythrocytes as revealed by a quick-freezing and deep-etching method. Virchows Arch [A] 422:73–80
Palek J, Sahr KE (1992) Mutation of the red blood cell membrane proteins: from clinical evaluation to detection of the underlying genetic defect. Blood 80:308–330
Palek J, Jarolim P (1993) Clinical expression and laboratory detection of red blood cell membrane protein mutations. Semin Hematol 30:249–283
Pothier B, Alloisio N, Maréchal J, Morlé L, Ducluzeau MT, Caldani C, Philipe N, Delaunay J (1990) Assignment of SpαI/74 hereditary elliptocytosis to the α- and β-chain of spectrin through in vitro dimer reconstitution. Blood 75:2061–2069
Sahr KE, Coetzer TL, Moy LS, Derick LH, Chishti AH, Jarolim P, Lorenzo F, Miraglia Del Giudice E, Iolascon A, Galanello R, Cao A, Delaunay J, Liu S-C, Palek J (1993) Spectrin Cagliari: an Ala to Gly substitution in helix 1 of β spectrin repeat 17 that severely disrupts the structure and self-association of the erythrocyte spectrin heterodimer. J Biol Chem 268:22656–22662
Shen BW, Josephs R, Steck TL (1986) Ultrastructure of the intra-skeleton of the erythrocyte membrane. J Cell Biol 102:997–1006
Smith SB, Revel JP (1972) Mapping of concanavalin A binding sites on the surface of several cell types. Dev Biol 27:434–441
Tse WT, Lecomte MC, Costa FF, Garbarz M, Féo C, Boivin P, Dhermy D, Forget BG (1990) Point mutation in the β-spectrin associated with αI/74 hereditary elliptocytosis. J Clin Invest 86:909–916
Tse WT, Gallagher PG, Pothier B, Costa FF, Scarpa A, Delaunay J, Forget BG (1991) An insertional frameshift mutation of the β-spectrin gene associated with elliptocytosis in spectrin Nice (β220/216). Blood 78:517–523
Tsukita S, Tsukita S, Ishikawa H (1980) Cytoskeletal network underlying the human erythrocyte membrane. J Cell Biol 85:567–576
Tyler JM, Hargreaves WR, Branton D (1979) Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1. Proc Natl Acad Sci USA 76:5192–5196
Ursitti JA, Wade JB (1993) Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton. Cell Motil Cytoskeleton 25:30–42
Ursitti JA, Pumplin DW, Wade JB, Bloch RJ (1991) Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane. Cell Motil Cytoskeleton 19:227–243
Weinstein RS, Tazelaar HD, Loew JM (1986) Ultrastructure of axial elongation of the membrane skeleton. Blood Cells 11:343–357
Yawata Y (1994) Red cell membrane protein band 4.2: phenotypic, genetic and electron microscopic aspects. Biochim Biophys Acta 1204:131–148
Yawata Y (1994) Band 4.2 abnormalities in human red cells. Am J Med Sci 307:190–203
Yoon SH, Yu H, Eber S, Prchal JT (1991) Molecular defect of truncated β-spectrin associated with hereditary elliptocytosis. β-Spectrin Göttingen. J Biol Chem 266:8490–8494
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Yawata, A., Kanzaki, A., Uehira, K. et al. A surface replica method: a useful tool for studies of the cytoskeletal network in red cell membranes of normal subjects and patients with a β-spectrin mutant (spectrin Le Puy: β220/214). Vichows Archiv A Pathol Anat 425, 297–304 (1994). https://doi.org/10.1007/BF00196153
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DOI: https://doi.org/10.1007/BF00196153