Aggregation of band 3 in hereditary ovalocytic red blood cell membranes. Electron microscopy and protein rotational diffusion studies

1993 ◽  
Vol 105 (3) ◽  
pp. 655-660
Author(s):  
A. Che ◽  
R.J. Cherry ◽  
L.H. Bannister ◽  
A.R. Dluzewski
Keyword(s):  
Electron Microscopy ◽  
Rotational Diffusion ◽  
Freeze Fracture ◽  
Band 3 ◽  
Normal Band ◽  
Cytoskeletal Proteins ◽  
Rotational Mobility ◽  
Normal Cells ◽  
Membrane Bound ◽  
Red Blood Cell Membranes

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G.B., Jones, G.L. and Sawyer, W.L. (1991) J. Membr. Biol. 121, 59–66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10–15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.

scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

scholarly journals Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2745-2753 ◽  
Author(s):  
AC Rybicki ◽  
RS Schwartz ◽  
EJ Hustedt ◽  
CE Cobb
Keyword(s):  
Correlation Time ◽  
Osmotic Fragility ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Erythrocyte Membranes ◽  
Rotational Mobility ◽  
Direct Role ◽  
Emission Anisotropy ◽  
Protein 4.2 ◽  
Nonionic Detergent

Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.

scholarly journals Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile

1997 ◽  
Vol 45 (10) ◽  
pp. 1365-1378 ◽  
Author(s):  
Moöse Bendayan ◽  
Eugenio A. Rasio
Keyword(s):  
Electron Microscopy ◽  
Serum Proteins ◽  
Protein A ◽  
Freeze Fracture ◽  
Thin Sections ◽  
Functional Studies ◽  
Transendothelial Transport ◽  
Rete Mirabile ◽  
Osmium Impregnation ◽  
Membrane Bound

The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular–vesicular system is involved in the transport of circulating proteins. Furthermore, protein A–gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular–vesicular system and is equivalent to the large pore system presumed from functional studies.

scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Abstract Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Freeze-Fracture Replication of Frozen Outer Segments of Rat Retinal Rods

1969 ◽  
Vol 27 ◽  
pp. 392-393
Author(s):  
Thomas S. Leeson ◽  
C. Roland Leeson
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Outer Segment ◽  
Freeze Fracture ◽  
X Ray Diffraction ◽  
X Ray ◽  
Outer Segments ◽  
Retinal Rods ◽  
Temperature Electron ◽  
Freeze Etching

Numerous previous studies of outer segments of retinal receptors have demonstrated a complex internal structure of a series of transversely orientated membranous lamellae, discs, or saccules. In cones, these lamellae probably are invaginations of the covering plasma membrane. In rods, however, they appear to be isolated and separate discs although some authors report interconnections and some continuities with the surface near the base of the outer segment, i.e. toward the inner segment. In some species, variations have been reported, such as longitudinally orientated lamellae and lamellar whorls. In cross section, the discs or saccules show one or more incisures. The saccules probably contain photolabile pigment, with resulting potentials after dipole formation during bleaching of pigment. Continuity between the lamina of rod saccules and extracellular space may be necessary for the detection of dipoles, although such continuity usually is not found by electron microscopy. Particles on the membranes have been found by low angle X-ray diffraction, by low temperature electron microscopy and by freeze-etching techniques.

Cryomicroscopy of multiphase latices

1991 ◽  
Vol 49 ◽  
pp. 1060-1061
Author(s):  
O. L. Shaffer ◽  
M.S. El-Aasser ◽  
C. L. Zhao ◽  
M. A. Winnik ◽  
R. R. Shivers
Keyword(s):  
Electron Microscopy ◽  
Radiation Damage ◽  
Freeze Fracture ◽  
Particle Morphology ◽  
Second Stage ◽  
Transmission Electron ◽  
Important Approach ◽  
Carbon Coated ◽  
Preparation Techniques

Transmission electron microscopy is an important approach to the characterization of the morphology of multiphase latices. Various sample preparation techniques have been applied to multiphase latices such as OsO4, RuO4 and CsOH stains to distinguish the polymer phases or domains. Radiation damage by an electron beam of latices imbedded in ice has also been used as a technique to study particle morphology. Further studies have been developed in the use of freeze-fracture and the effect of differential radiation damage at liquid nitrogen temperatures of the latex particles embedded in ice and not embedded.Two different series of two-stage latices were prepared with (1) a poly(methyl methacrylate) (PMMA) seed and poly(styrene) (PS) second stage; (2) a PS seed and PMMA second stage. Both series have varying amounts of second-stage monomer which was added to the seed latex semicontinuously. A drop of diluted latex was placed on a 200-mesh Formvar-carbon coated copper grid.

Quantitative Freeze Fracture Studies of Gap Junctions in Somatic Cell Hybrids, Their Parents, and Revertants

1976 ◽  
Vol 34 ◽  
pp. 218-219
Author(s):  
W. J. Larsen ◽  
R. Azarnia ◽  
W. R. Loewenstein
Keyword(s):  
Gap Junctions ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Cell Movement ◽  
Dye Molecules ◽  
Somatic Cell Hybrids ◽  
Cell Coupling ◽  
Normal Cells ◽  
Mediate Cell ◽  
Almost All

Although the physiological significance of the gap junction remains unspecified, these membrane specializations are now recognized as common to almost all normal cells (excluding adult striated muscle and some nerve cells) and are found in organisms ranging from the coelenterates to man. Since it appears likely that these structures mediate the cell-to-cell movement of ions and small dye molecules in some electrical tissues, we undertook this study with the objective of determining whether gap junctions in inexcitable tissues also mediate cell-to-cell coupling.To test this hypothesis, a coupling, human Lesh-Nyhan (LN) cell was fused with a non-coupling, mouse cl-1D cell, and the hybrids, revertants, and parental cells were analysed for coupling with respect both to ions and fluorescein and for membrane junctions with the freeze fracture technique.

Ultrastructural Features of Normal and Diseased Hair Revealed by Ion Beam Etching and Freeze Fracture

1975 ◽  
Vol 33 ◽  
pp. 358-359
Author(s):  
M. Spector ◽  
A. C. Brown
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Ectodermal Dysplasia ◽  
Ion Beam ◽  
Freeze Fracture ◽  
Ion Current ◽  
Ion Beam Etching ◽  
Pili Torti ◽  
Argon Ion ◽  
Scanning Electron

Ion beam etching and freeze fracture techniques were utilized in conjunction with scanning electron microscopy to study the ultrastructure of normal and diseased human hair. Topographical differences in the cuticular scale of normal and diseased hair were demonstrated in previous scanning electron microscope studies. In the present study, ion beam etching and freeze fracture techniques were utilized to reveal subsurface ultrastructural features of the cuticle and cortex.Samples of normal and diseased hair including monilethrix, pili torti, pili annulati, and hidrotic ectodermal dysplasia were cut from areas near the base of the hair. In preparation for ion beam etching, untreated hairs were mounted on conducting tape on a conducting silicon substrate. The hairs were ion beam etched by an 18 ky argon ion beam (5μA ion current) from an ETEC ion beam etching device. The ion beam was oriented perpendicular to the substrate. The specimen remained stationary in the beam for exposures of 6 to 8 minutes.

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