Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane

Ursitti, Jeanine A.; Pumplin, David W.; Wade, James B.; Bloch, Robert J.

doi:10.1002/cm.970190402

Cited by 93 publications

(93 citation statements)

References 54 publications

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“…The tortuous trajectory of minimally perturbed erythroid spectrin in situ imaged by electron microscopy (45), and later confirmed by atomic force microscopy of dried (46) and hydrated (47,48) red cells, implies that spectrin can bend in a nonuniform fashion along its length. Likewise, rotary-shadowed human (49) and avian (50) spectrin dimers and tetramers exhibit different degrees of curvature, although not consistently in the same regions of the molecules.…”

Section: Evidence For Varying Flexibility Along the Spectrin Tetramermentioning

confidence: 87%

Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer

MacDonald

Cummings²

2004

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

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The large size of spectrin, the flexible protein promoting reversible deformation of red cells, has been an obstacle to elucidating the molecular mechanism of its function. By studying cloned fragments of the repeating unit domain, we have found a correspondence between positions of selected spectrin repeats in a tetramer with their stabilities of folding. Six fragments consisting of two spectrin repeats were selected for study primarily on the basis of the predicted secondary structures of their linker regions. Fragments with a putatively helical linker were more stable to urea-and heat-induced unfolding than those with a putatively nonhelical linker. Two of the less stably folded fragments, human erythroid ␣-spectrin repeats 13 and 14 (HE␣13,14) and human erythroid ␤-spectrin repeats 8 and 9 (HE␤8,9), are located opposite each other on antiparallel spectrin dimers. At least partial unfolding of these repeats under physiological conditions indicates that they may serve as a hinge. Also less stably folded, the fragment of human erythroid ␣-spectrin repeats 4 and 5 (HE␣4,5) lies opposite the site of interaction between the partial repeats at the C-and N-terminal ends of ␤-and ␣-spectrin, respectively, on the opposing dimer. More stably folded fragments, human erythroid ␣-spectrin repeats 1 and 2 (HE␣1,2) and human erythroid ␣-spectrin repeats 2 and 3 (HE␣2,3), lie nearly opposite each other on antiparallel spectrin dimers of a tetramer. These clusterings along the spectrin tetramer of repeats with similar stabilities of folding may have relevance for spectrin function, particularly for its well known flexibility.

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Section: Evidence For Varying Flexibility Along the Spectrin Tetramermentioning

confidence: 87%

Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer

MacDonald

Cummings²

2004

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

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“…First, the PI(4,5)P 2 concentration in the PM has been determined using erythrocytes (11,20,28). These cells have a unique cytoskeleton structure (5,27,86) and lack the regular endocytosis function (77). As actin cytoskeleton rearrangement and endocytosis are two major PI(4,5)P 2 -regulated cellular functions in other cell types (13,47,49,85), PI(4,5)P 2 levels in erythrocytes may differ from those in other cell types.…”

Section: Discussionmentioning

confidence: 99%

Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

Chukkapalli

Hogue

Boyko

et al. 2008

J Virol

250

386

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Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P 2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P 2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P 2 -depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P 2 . To examine a putative Gag interaction with PI(4,5)P 2 , we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P 2 . Using this assay, we observed that PI(4,5)P 2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P 2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P 2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P 2 -containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P 2 on the membrane and that the MA basic domain mediates this interaction.

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“…In addition, if the cytoskeletal network serves as a physical barrier, in order to account for a retardation in entry of a factor of 800 (36) this mechanism would require that such a network would cover all but 1/800 ϭ 0.125% of the total inner surface area of the membrane. The spectrin component is an elongated rod that forms a web with large spaces inbetween (37). As suggested by Huang et al (36), hemoglobin could bind to the membrane and conceivably cover parts of the surface.…”

Section: The Effect Of Hematocrit On the Flux Of No Transfer Intomentioning

confidence: 99%

Nitric Oxide Uptake by Erythrocytes Is Primarily Limited by Extracellular Diffusion Not Membrane Resistance

Liu¹,

Samouilov²,

Lancaster³

et al. 2002

Journal of Biological Chemistry

114

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The process of NO transfer into erythrocytes (RBCs) is of critical biological importance because it regulates the bioavailability and diffusional distance of endothelialderived NO. It has been reported that the rate of NO reaction with oxyhemoglobin (Hb) within RBCs is nearly three orders of magnitude slower than that by equal amounts of free oxyhemoglobin. Consistent with early studies on oxygen uptake by RBCs, the process of extracellular diffusion was reported to explain this much lower NO uptake by RBC encapsulated Hb (Liu, X., Miller, M. J., Joshi, M. S., Sadowska-Krowicka, H., Clark, D. A., and Lancaster, J. R., Jr. (1998) J. Biol. Chem. 273, 18709 -18713). However, it was subsequently proposed that the RBC membrane provides the main resistance to NO uptake rather than the process of extracellular diffusion (Vaughn, M. W., Huang, K. T., Kuo, L., and Liao, J. C. (2000) J. Biol. Chem. 275, 2342-2348). This conclusion was based on competition experiments that were assumed to be able to determine the rate constant of NO uptake by RBCs without extracelluar diffusion limitation. To test the validity of this hypothesis, we theoretically analyzed competition experiments. Here, we show that competition experiments do not eliminate the extracellular diffusion limitation. Simulation of the competition data indicates that the main resistance to NO uptake by RBCs is caused by extracellular diffusion in the unstirred layer surrounding each RBC but not by the RBC membrane. This extracellular diffusion resistance is responsible for preventing interference of NO signaling in the endothelium without the need for special NO uptake by intracellular hemoglobin or a unique membrane resistance mechanism.Nitric oxide (nitrogen monoxide, NO) has been recognized as a critical physiological mediator in the regulation of vascular tone (1). It is generated in vascular endothelium by a specific arginine-dependent NO synthase, NOS3. Free diffusion has been considered to be the main process that determines how NO travels from the site of its formation to target sites where it exerts its critical physiological functions (2, 3).In the vascular lumen NO can be scavenged by hemoglobin subunits (Hb) 1 within RBCs. The reaction of NO with myoglobin and hemoglobin is rapid (bimolecular rate constant for oxyMb is k Mb ϭ 3 ϫ 10 7 M Ϫ1 s Ϫ1 and for oxyHb is k Hb ϭ 6-9 ϫ 10 7 M Ϫ1 s Ϫ1 ) (4, 5). It has been reported that 10 M cell-free Hb is enough to trap almost all NO production generated from endothelial cells and abolish NO-mediated vasodilation (6, 7), while blood contains about 8 mM Hb, which is much higher than the quantity of Hb required to completely eliminate NO-mediated functional effects. The rapid reaction between NO and oxyhemoglobin raises the question of how NO can escape from the large quantity of Hb in the blood vessel lumen to exert physiological effects in the blood vessel wall after it is generated from endothelial cells (2,8). This has led to recent efforts to understand and theoretically model the reaction process between NO ...

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Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane

Cited by 93 publications

References 54 publications

Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer

Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer

Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

Nitric Oxide Uptake by Erythrocytes Is Primarily Limited by Extracellular Diffusion Not Membrane Resistance

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