scholarly journals Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

1980 ◽  
Vol 86 (2) ◽  
pp. 694-698 ◽  
Author(s):  
C M Cohen ◽  
S F Foley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Actin Filaments ◽  
Binding Capacity ◽  
Protein Band ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Cytoplasmic Surface ◽  
Independent Association ◽  
Inside Out

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

scholarly journals Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

1981 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
S Sato ◽  
M Nakao
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Thin Sections ◽  
Fixation Treatment ◽  
Human Erythrocyte Membranes ◽  
Inside Out

Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid- glutaraldehyde fixation. Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted more than 90 percent of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles. When such spectrin-depleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles. The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form a irregular but continuous meshwork parallel to the membrane. Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles. However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin- or purified spectrin- reassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components. Two modes of association of actin filaments with the membrane were seen: end-to-membrane and side-to- membrane associations. In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane.

scholarly journals Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

1981 ◽  
Vol 88 (2) ◽  
pp. 388-395 ◽  
Author(s):  
VM Fowler ◽  
EJ Luna ◽  
WR Hargreaves ◽  
DL Taylor ◽  
D Branton
Keyword(s):  
Erythrocyte Membrane ◽  
Attachment Site ◽  
Water Soluble ◽  
Actin Binding ◽  
Cytoplasmic Surface ◽  
Multiple Binding Sites ◽  
Affinity Membrane ◽  
Solution Studies ◽  
Membrane Attachment ◽  
Inside Out

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

scholarly journals Hereditary elliptocytosis with protein band 4.1 deficiency in the dog

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 373-377 ◽  
Author(s):  
JE Smith ◽  
K Moore ◽  
M Arens ◽  
GA Rinderknecht ◽  
A Ledet
Keyword(s):  
Animal Model ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Deformability ◽  
Membrane Stability ◽  
Osmotic Sensitivity ◽  
Hereditary Elliptocytosis ◽  
Membrane Cytoskeleton ◽  
Glutathione Deficiency

Abstract A dog with persistent elliptocytosis was studied. The dog had membrane protein band 4.1 deficiency, microcytosis, shortened erythrocyte lifespan, increased osmotic sensitivity, and a mild glutathione deficiency. Erythrocyte deformability and membrane stability were adversely effected. The dog's parents had decreased band 4.1, decreased stability, and some elliptocytosis. This disorder in dogs closely resembles human patients with band 4.1 deficiency and should provide a valuable animal model to study the erythrocyte membrane cytoskeleton.

Nuclear ion channel activity is regulated by actin filaments

1996 ◽  
Vol 270 (5) ◽  
pp. C1532-C1543 ◽  
Author(s):  
A. G. Prat ◽  
H. F. Cantiello
Keyword(s):  
Ion Channels ◽  
Actin Cytoskeleton ◽  
Nuclear Envelope ◽  
Ion Channel ◽  
Actin Filaments ◽  
Actin Binding ◽  
Bathing Solution ◽  
Channel Activity ◽  
Nuclear Ion Channels ◽  
Inside Out

Actin filaments are novel second messengers involved in ion channel regulation. Because cytoskeletal components interact with the nuclear envelope, the actin cytoskeleton may also control nuclear membrane function. In this report, the patch-clamp technique was applied to isolated nuclei from amphibian A6 epithelial cells to assess the role of actin filaments on nuclear ion channel activity under nucleus-attached or -excised conditions. The most prevalent spontaneous nuclear ion channel species, 76% (n = 46), was cation selective and had a maximal single-channel conductance of approximately 420 pS. Nuclear ion channels also displayed multiple subconductance states, including channel activity of 26 pS that was frequently observed. Nuclear ion channel activity on otherwise quiescent patches was induced by either addition of the actin cytoskeleton disrupter cytochalasin D (CD; 5 micrograms/ml, 60%, 3 of 5 patches) or actin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches (59%, 13 of 22 patches). Actin also induced ion channel activity in quiescent excised inside-out patches from the nuclear envelope (80%, 4 of 5 patches). In contrast, addition of bovine serum albumin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches was without effect on nuclear ion channel activity (5 of 5 patches). The monoclonal antibody MAb414, specific for nuclear pore complex proteins, completely prevented either spontaneous or cytosolic actin-induced nuclear ion channels under nucleus-attached conditions (4 of 4 patches) but not intranuclear actin-induced nuclear ion channels under excised inside-out conditions (3 of 3 patches). In nucleus-attached patches, channel activity was readily activated by addition of the G-actin-binding protein deoxyribonuclease I to nucleus-attached patches (56%, 5 of 9 patches) or further addition of the actin-cross-linker filamin in the presence of actin (57%, 4 of 7 patches). The data indicate that dynamic changes in actin filament organization may represent a novel mechanism to control nuclear function.

scholarly journals Bidirectional polymerization of G-actin on the human erythrocyte membrane.

1984 ◽  
Vol 98 (3) ◽  
pp. 1102-1110 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa
Keyword(s):  
Erythrocyte Membrane ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Experimental System ◽  
Opposite Polarity ◽  
Erythrocyte Membranes ◽  
Low Concentrations ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Almost All

The directional polymerization of actin on the erythrocyte membrane has been examined at various concentrations of G-actin by thin-section electron microscopy. For this purpose, a new experimental system using single-layered erythrocyte membranes with the cytoplasmic surfaces freely exposed was developed. The preformed actin filaments did not bind with the cytoplasmic surface of the erythrocyte membranes. When the erythrocyte membranes were incubated at low concentrations (0.3 and 0.5 microM) of G-actin, greater than 80% of polymerized actin filaments pointed toward the membranes mainly in an end-on fashion, as judged by arrowhead formation with heavy meromyosin. At higher concentrations (2 and 4 microM) of G-actin, about half of the polymerized actin filaments were directed with arrowheads pointing toward the membranes, while the rest of the filaments showed the opposite polarity pointing away from the membranes. The majority of polymerized actin filaments formed loops at the points of attachment to the membranes. In contrast, when G-actin (2 and 4 microM) in the presence of cytochalasin B was polymerized into filaments, approximately 70% showed the polarity pointing away from the membrane mainly in an end-on fashion. To check the treadmilling phenomena, the erythrocyte membranes with bidirectionally polymerized actin filaments were further incubated with G-actin at the overall critical concentration. In this case, almost all (90%) of actin filaments showed the polarity with arrowheads pointing toward the membranes. The results obtained are discussed with special reference to the mode of association of actin filaments with the plasma membrane in general.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes

1978 ◽  
Vol 56 (5) ◽  
pp. 349-351 ◽  
Author(s):  
J. Thomas Buckley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Lipid Composition ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Firm Conclusion ◽  
Membrane Phospholipids ◽  
Human Erythrocyte Membranes ◽  
Significant Enrichment

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparations. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium diiodosalicylate are recovered in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.

Membrane orientation of sheep spectrin

1980 ◽  
Vol 58 (10) ◽  
pp. 1120-1130
Author(s):  
P. Prokopchuk ◽  
A. U. Sargent
Keyword(s):  
Sheep Erythrocyte ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Variable Degree ◽  
Vertebrate Species ◽  
Membrane Orientation ◽  
Cytoplasmic Surface ◽  
Immunofluorescence Studies ◽  
Surface Labelling ◽  
Inside Out

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membranes localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected i0mmunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.

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