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 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.

Evidence for an Asymmetric Distribution of Phospholipids in the Human Erythrocyte Membrane

1974 ◽  
Vol 52 (9) ◽  
pp. 803-806 ◽  
Author(s):  
Arthur Kahlenberg ◽  
Caroline Walker ◽  
Ruth Rohrlick
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Phospholipid Composition ◽  
Enzyme Hydrolysis ◽  
Asymmetric Distribution ◽  
Human Erythrocyte Membrane ◽  
Cytoplasmic Surface ◽  
Direct Demonstration ◽  
Inside Out ◽  
Phospholipases A

The changes in phospholipid composition of the inner (cytoplasmic) surface of the human erythrocyte membrane resulting from the digestion of sealed inside-out vesicles with phospholipases A2 and C were determined. Virtually all of the phosphatidylethanolamine and phosphatidylserine and 30–40% of the phosphatidylcholine and sphingomyelin of inside-out vesicles were found to be accessible to enzyme hydrolysis. In contrast, all of the above phospholipids of unsealed ghosts were susceptible to phospholipolytic digestion. These results are a direct demonstration of an asymmetric distribution of phospholipids in the human erythrocyte membrane.

scholarly journals Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 29-43 ◽  
Author(s):  
EA Evans ◽  
PL La Celle
Keyword(s):  
Surface Area ◽  
Plastic Flow ◽  
Erythrocyte Membrane ◽  
Permanent Deformation ◽  
Volume Ratio ◽  
Total Surface Area ◽  
Water Soluble ◽  
Time Range ◽  
Extrinsic Factors ◽  
Red Cell Membrane

Abstract Deformation of the erythrocyte membrane by the micropipette technique permits analysis of intrinsic material characteristics of the membrane and provides a means to differentiate purely membrane factors from such extrinsic factors as surface area-to-volume ratio. Using small micropipettes (less than 0.5 microns radius) to deform cells, it is evident that the red cell membrane behaves like a solid for periods of time up to 5–10 min of sustained deformation; for long periods of strain, permanent deformations occur, indicative of the semi-solid structural character. In the time range in which the membrane behaves like a solid, the material is linearly elastic up to strains of 400%, implying a loose network structure in the membrane plane, and evaluation of the elastic parameter mu (mu for normal discocytes equals 7 x 10(-3) dynes/cm) suggests that the elements comprising the network may have a molecular weight of approximately that of the water-soluble membrane protein spectrin. Whether the network system is cross-linked or simply a polymer solution remains unanswered. Experimental data indicate that plastic flow of the membrane under conditions of protracted strain may lead to permanent deformation of the membrane, whereas uniform dilation of the membrane, requiring over 1000 times more energy than for plastic flow, results in membrane failure and lysis. Analyses of the data from larger micropipettes of limiting mean cylindrical diameter show their utility in evaluating extrinsic factors, e.g., surface area-to-volume relationships, which are related to the capability of the whole cell to form a new configuration with implicit resistance to total surface area change, as the cell enters narrow channels of the microcirculation. Thus, micropipettes with diameters in the 2.7–3.0-microns range can provide sensitive comparisons of cellular deformability of erythrocytes.

scholarly journals Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase of Thermus thermophilus

2005 ◽  
Vol 187 (12) ◽  
pp. 3990-3996 ◽  
Author(s):  
Olga Zafra ◽  
Felipe Cava ◽  
Francis Blasco ◽  
Axel Magalon ◽  
Jose Berenguer
Keyword(s):  
Nitrate Reductase ◽  
Thermus Thermophilus ◽  
Attachment Site ◽  
Soluble Form ◽  
Strong Interactions ◽  
E Coli ◽  
Membrane Complex ◽  
Cytoplasmic Face ◽  
Membrane Attachment ◽  
Mature Enzyme

ABSTRACT The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

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 Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.

1978 ◽  
Vol 76 (2) ◽  
pp. 512-531 ◽  
Author(s):  
D Shotton ◽  
K Thompson ◽  
L Wofsy ◽  
D Branton
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Indirect Evidence ◽  
Surface Proteins ◽  
Human Erythrocyte Membrane ◽  
Intramembrane Particle ◽  
Translational Movement ◽  
Cytoplasmic Surface ◽  
Particle Aggregates ◽  
Before And After

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

Dematin and Adducin Provide a Critical Link between the Spectrin Cytoskeleton and the Erythrocyte Membrane Via Glucose Transporter-1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1719-1719
Author(s):  
Anwar A. Khan ◽  
Toshihiko Hanada ◽  
Massimiliano Gaetani ◽  
Donghai Li ◽  
Brent C. Reed ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Erythrocyte Membrane ◽  
Glucose Transporter ◽  
Transmembrane Protein ◽  
Actin Binding ◽  
Junctional Complex ◽  
Head Region ◽  
Glucose Transporter 1 ◽  
Erythrocyte Shape ◽  
Spectrin Cytoskeleton

Abstract There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

Conditional Knockout-First Gene Disruption of Dematin Causes Precipitous Loss of Erythrocyte Membrane Stability and Severe Hemolytic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 157-157
Author(s):  
Yunzhe Lu ◽  
Toshihiko Hanada ◽  
Athar H. Chishti
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Hemolytic Anemia ◽  
Erythrocyte Membrane ◽  
Half Life ◽  
Gene Disruption ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Wild Type

Abstract Dematin is an actin binding and bundling protein originally identified as a component of the erythrocyte membrane junctional complex. A widely expressed member of the villin-family of adaptor proteins, dematin regulates RhoA activity and cell shape in fibroblasts. Actin binding and bundling activity of dematin is regulated by phosphorylation of its headpiece domain by the cAMP-dependent protein kinase. Despite its extensive biochemical characterization, the physiological function of dematin in mature erythrocytes remains unknown. We used a conditional gene disruption strategy by generating a targeting construct that has the potential for full body gene knockout as well as tissue-specific deletion of dematin gene using the Cre-lox gene deletion system. Wild type, heterozygous, and homozygous progeny were obtained in a typical Mendelian ratio of 1:2:1. Dramatic splenomegaly in 7-week old full length dematin knockout (FLKO) mice was observed with the average spleen weight 10-fold higher than those of the wild type littermates. Flow cytometry showed a ~16-fold increase in reticulocytes (Fig.1A), which was also seen in the blood smear (Fig.1B,C). Severe hemolytic anemia is most likely the cause of relative pallor observed in FLKO mice at day 1 after birth. The adult FLKO mice continue to show relatively smaller body size as compared to wild type and heterozygous mice. These findings are consistent with severe anemia and compensatory erythropoiesis. FLKO mice exhibit typical signs of anisocytosis, microcytosis, macrocytosis, and polychromasia, which are indicative of tremendous variation in RBC cell size and the premature release of reticulocytes from the bone marrow. Moreover, additional RBC abnormalities, including poikilocytosis, acanthocytosis, fragmented RBC, and spherocytes, are consistent with severe hemolytic disease. By scanning EM, the FLKO erythrocytes showed dramatic variation in shape and size. The spherocytes, microcytic vesiculation, and the protruding structures are observed in FKLO mice, as well as extensive intravascular hemolysis (Fig. 1D,E). RBC half-life measurements in vivo by NHS-biotin labeling and flow cytometry showed mutant cells almost immediately cleared from the circulation in FLKO mice. A seven-week chase experiment showed that the half-life of RBCs was reduced from 22 days in wild type and heterozygous mice to less than 3 days in FLKO mice. The hematological phenotype of FLKO mice indicated reduced RBC count, hemoglobin, and hematocrit with increase in the RBC distribution width. Collectively, these findings indicate that the mechanical strength of RBC membrane strictly relies on the presence of full length dematin. We employed membrane fractionation, in vitro protein domain mapping, transmission/scanning electron microscopy, and dynamic deformability measurements to investigate the underlying mechanisms of extreme membrane fragility in FLKO erythrocytes. We also examined the protein profile of RBC ghosts. Surprisingly, the major cytoskeletal proteins remained unchanged in the FLKO ghosts; however, a marked reduction of spectrin, adducin, and actin was observed. When normalized against band 3, these proteins were reduced by 60%, 90%, and 90%, respectively. Since these membrane proteins are essential for RBC stability, our findings suggest a specific role of dematin in recruiting or maintaining a stable association of essential cytoskeletal proteins in the plasma membrane. These results raise the possibility that dematin may directly interact with adducin, and together anchor the spectrin molecules to the plasma membrane. Our findings provide the first in vivo evidence that dematin is essential for the maintenance of erythrocyte shape and membrane mechanical properties by regulating the integrity of the spectrin-actin junctions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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