Red Blood Cell Cytoskeleton Research Articles

Tether Extrusion from Red Blood Cells: Integral Proteins Unbinding from Cytoskeleton

We investigate the mechanical strength of adhesion and the dynamics of detachment of the membrane from the cytoskeleton of red blood cells (RBCs). Using hydrodynamical flows, we extract membrane tethers from RBCs locally attached to the tip of a microneedle. We monitor their extrusion and retraction dynamics versus flow velocity (i.e., extrusion force) over successive extrusion-retraction cycles. Membrane tether extrusion is carried out on healthy RBCs and ATP-depleted or -inhibited RBCs. For healthy RBCs, extrusion is slow, constant in velocity, and reproducible through several extrusion-retraction cycles. For ATP-depleted or -inhibited cells, extrusion dynamics exhibit an aging phenomenon through extrusion-retraction cycles: because the extruded membrane is not able to retract properly onto the cell body, each subsequent extrusion exhibits a loss of resistance to tether growth over the tether length extruded at the previous cycle. In contrast, the additionally extruded tether length follows healthy dynamics. The extrusion velocity L ˙ depends on the extrusion force f according to a nonlinear fashion. We interpret this result with a model that includes the dynamical feature of membrane-cytoskeleton association. Tether extrusion leads to a radial membrane flow from the cell body toward the tether. In a distal permeation regime, the flow passes through the integral proteins bound to the cytoskeleton without affecting their binding dynamics. In a proximal sliding regime, where membrane radial velocity is higher, integral proteins can be torn out, leading to the sliding of the membrane over the cytoskeleton. Extrusion dynamics are governed by the more dissipative permeation regime: this leads to an increase of the membrane tension and a narrowing of the tether, which explains the power law behavior of L ˙ ( f ) . Our main result is that ATP is necessary for the extruded membrane to retract onto the cell body. Under ATP depletion or inhibition conditions, the aging of the RBC after extrusion is interpreted as a perturbation of membrane-cytoskeleton linkage dynamics.

General up to next-nearest-neighbour elasticity of triangular lattices in three dimensions

We establish the most general form of the discrete elasticity of a two-dimensional triangular lattice embedded in three dimensions, taking into account up to next-nearest-neighbour interactions. Besides crystalline system, this is relevant to biological physics (e.g. red blood cell cytoskeleton) and soft matter (e.g. percolating gels, etc.). In order to correctly impose the rotational invariance of the bulk terms, it turns out to be necessary to take into account explicitly the elasticity associated with the vertices located at the edges of the lattice. We find that some terms that were suspected in the literature to violate rotational symmetry are, in fact, admissible.

Conformational Changes in the Cytoplasmic Domain of Human Anion Exchanger 1 Revealed by Luminescence Resonance Energy Transfer

The cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) serves as a center of organization for the red blood cell cytoskeleton as well as several metabolic enzymes and hemoglobin. The protein is known to undergo a reversible pH-dependent conformational change characterized by a 2-fold change in the intrinsic fluorescence and an 11 A change in the Stokes radius. While the exact changes in the molecular structure are unknown, on the basis of the crystal structure of the protein at pH 4.8 and site-directed mutagenesis studies, Zhou and Low (19) have proposed that the peripheral protein binding (PPB) domain of cdAE1 moves away from the dimerization domain in response to increasing alkalinity. To test this hypothesis, we have applied luminescence resonance energy transfer (LRET) to measure the intermonomer distance between donor and acceptor probes at the Cys201 site (located in the PPB domain) within the cdAE1 dimer. This distance was found to increase as the pH is increased from 5 to 10, in recombinant forms of both the wild type and a mutant (C317S) of cdAE1. Furthermore, LRET measurements in red blood cell inside-out vesicles indicate that when cdAE1 is linked to the membrane, the intermonomer distance is larger at pH 5, compared to that of the purified cdAE1 segments, and exhibits a different pH-dependent behavior. An increase in the distance was also observed on binding of a metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, to cdAE1. These data provide the first demonstration of a defined change in the molecular structure of cdAE1, and also indicate that the structure under physiological conditions is different from the crystal structure determined at low pH.

Erythrocyte Cytoskeletal Defects Induced in Mice by Deletion of Rac GTPases.

The small Rho GTPases Rac1 and Rac2 have been implicated in regulating actin structures in a variety of cells, including hematopoietic stem cells and leucocytes. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. We explored the possible role of Rac1 and Rac2 signaling molecules in the dynamic assembly of actin in the red blood cells (RBC), and thus in the regulation of morphology and function of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases have been shown to have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in hematopoietic stem cells (Gu et al. Science, 2003); we focused our study on the erythrocyte phenotype of Rac2−/− and Rac1−/−;Rac2−/− mice. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background. Deletion of Rac1 after treatment with PolyI:PolyC to induce Cre recombinase was confirmed in bone marrow cells using DNA PCR and in erythrocytes by immunoblot. Since the erythrocytes consist a population of variable age, the optimal time of the maximum Rac1 deletion in erythrocytes was determined to be three to five weeks post induction. During this period, Rac1 protein in erythrocytes was decreased by 50–80% as determined by immunoblot densitometry. Rac2−/− and wild-type mice were subjected to the same treatment to control for any effects of PolyI:PolyC independent of the Rac1 deletion. Blood samples were obtained weekly after the completion of induction and the hematologic phenotype was studied by evaluation of complete blood counts, RBC indices, and reticulocyte counts. Erythrocyte morphology was examined on Wright-Giemsa smears of peripheral blood. Intact erythrocytes and erythrocyte ghosts were stained for actin with rhodamine-phalloidin and studied by confocal microscopy. The Rac2−/− mice appeared to have a rather mild erythrocyte phenotype with no significant anemia or reticulocytosis, although they did demonstrate a mild poikilocytosis and anisocytosis at baseline. The Rac1−/−;Rac2−/− mice developed a microcytic anemia with a hemoglobin drop of up to 30% in comparison to the baseline and to the wild-type hemoglobin values, with the nadir noted at three weeks post induction. The percentage of reticulocytes increased up to threefold in comparison to the control group. The mean corpuscular volume decreased up to 20% from the baseline in the Rac1−/−;Rac2−/− mice, and remained decreased up to six weeks post induction with an elevated red blood cell distribution width. Significant anisocytosis and poikilocytosis were observed with fragmented erythrocytes in the peripheral blood smear. Filamentous actin in the RBC cytoskeleton stained with rhodamine-phalloidin appeared to have a uniform distribution in intact and ghost erythrocytes under confocal microscopy. However, Rac1−/−;Rac2−/− erythrocytes demonstrated punctuate lesions on the cell surface while Rac1−/−;Rac2−/− erythrocyte ghosts appeared to collapse into irregular shapes. These data suggest that deficiency of Rac1 and Rac2 GTPases in mice cause a microcytic hemolytic anemia with poikilocytosis and red cell fragmentation indicating a possible dynamic regulation of the erythrocyte cytoskeleton organization by these signaling molecules.

Mutations in the murine erythroid α-spectrin gene alter spectrin mRNA and protein levels and spectrin incorporation into the red blood cell membrane skeleton

AbstractTetramers of α- and β-spectrin heterodimers, linked by intermediary proteins to transmembrane proteins, stabilize the red blood cell cytoskeleton. Deficiencies of either α- or β-spectrin can result in severe hereditary spherocytosis (HS) or hereditary elliptocytosis (HE) in mice and humans. Four mouse mutations,sph,sphDem,sph2BC, andsphJ, affect the erythroid α-spectrin gene,Spna1, on chromosome 1 and cause severe HS and HE. Here we describe the molecular alterations in α-spectrin and their consequences insph2BC/sph2BCandsphJ/sphJerythrocytes. A splicing mutation,sph2BCinitiates the skipping of exon 41 and premature protein termination before the site required for dimerization of α-spectrin with β-spectrin. A nonsense mutation in exon 52,sphJeliminates the COOH-terminal 13 amino acids. Both defects result in instability of the red cell membrane and loss of membrane surface area. Insph2BC/sph2BC, barely perceptible levels of messenger RNA and consequent decreased synthesis of α-spectrin protein are primarily responsible for the resultant hemolysis. By contrast,sphJ/sphJmice synthesize the truncated α-spectrin in which the 13-terminal amino acids are deleted at higher levels than normal, but they cannot retain this mutant protein in the cytoskeleton. ThesphJdeletion is near the 4.1/actin-binding region at the junctional complex providing new evidence that this 13-amino acid segment at the COOH-terminus of α-spectrin is crucial to the stability of the junctional complex.

Modulation of nitric oxide bioavailability by erythrocytes.

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs.

Immunoreactivity of Common Red Blood Cell Antigens in Cytoskeleton-Free Red Blood Cell Microvesicles

Background Cytoskeleton-free microvesicles can be generated from normal red blood cells (RBCs). These RBC vesicles maintain a representative sampling of the lipid bilayer and several membrane proteins. We investigated RBC antigen segregation and persistence of immunoreactivity in RBC microvesicles. Methods Cytoskeleton-free RBC microvesicles were generated from erythrocytes expressing known RBC antigens. Antigen segregation into vesicles was documented by immunospecific antigen-antibody binding using IgG eluates and 125I Protein A. 125I Protein A counts per minute (cpm) ratios between antigen-positive vesicles sensitized with 11 eluates compared with those of vesicles incubated with normal human serum are reported. Results Cytoskeleton-free RBC microvesicles preserved the antigenic expression of the original RBCs. Differences in cpm between eluate-sensitized vesicles and those incubated with normal human serum ranged from 3:1 for the Fy b antigen to 65:1 for the D antigen. Conclusions This study demonstrates that molecules bearing common RBC antigens segregate into microvesicles, fully preserving their epitope-bearing activity. The major proteins of the RBC cytoskeleton are not required for such antigen expression.

Ghost protein damage by peroxynitrite and its protection by melatonin.

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Renal Abnormalities at the Prehypertensive Stage of Essential Hypertension

An approach to evaluating the genetic components of essential hypertension using an animal model, the Milan hypertensive strain (MHS) of rats, and studies in human families with positive and negative histories for high blood pressure are described and discussed. Differences at renal and cellular levels between MHS and its normotensive control strain, MNS, show many similarities to those between offspring from hyper- and normotensive families in humans. These include, with respect to the former group of each species, lower erythrocyte volume and Na content, higher Na-K cotransport across red blood cell (RBC) membranes higher Na excretion after load, and greater pressor effect in transplanted kidneys. A novel protein found in rat RBC cytoskeleton appears to have a function in Na-K cotransport and it may, eventually, be possible to demonstrate in man.

In vitro phosphorylation of the red blood cell cytoskeleton complex by cyclic AMP-dependent protein kinase from erythrocyte membrane

Hydrosoluble proteins extracted from human erythrocyte ghosts by dialysis at low ionic strength and alkaline pH contain a cyclic AMP-dependent protein kinase which phosphorylates in vitro the cytoskeleton components in crude extracts. Spectrin components 1 and 2, actin and protein band 4.1 undergo this cyclic AMP-dependent endogenous phosphorylation together with low molecular weight peptides solubilized with the cytoskeleton in hydrosoluble extract. However, pure spectrin and purified erythrocyte G-actin were not phosphorylated by purified cyclic AMP-dependent protein kinase from erythrocyte membrane. Purified G-actin when added free to crude extract does not undergo phosphorylation by the cyclic AMP-dependent protein kinase present in this extract. In contrast, purified cyclic AMP-dependent protein kinase added either to crude extract or to the purified cytoskeleton complex (spectrin, actin and protein band 4.1), phosphorylates spectrin, actin and protein band 4.1. We can conclude that (1) cyclic AMP-dependent phosphorylation of red cell cytoskeleton occurs in vitro only when the cytoskeleton components are in a complexed form; (2) red cell actin, like other cellular actins, may be phosphorylated by cyclic AMP-dependent protein kinase but only in the oligomeric form and not in the G form.

Influence of red cell shape on surface charge topography

Human red blood cells (RBCs), transformed from discocytes into crenated shapes by incubation in hyperosmotic NaCl, have increased rates of agglutination in the presence of either poly- l-lysine (PLL) or soybean agglutinin (SBA). Reversion to discocytes by resuspension in isoosmotic NaCl lowers agglutination rates back to normal levels. Crenation also causes a reversible decrease in the binding of cationized ferritin (CF) particles/μm 2 of RBC surface area and focal clustering of CF particles. The decrease in CF binding and focal CF clustering are probably due to a rearrangement of negative charge bearing molecules such as glycoproteins on the RBC surface. Clustering of membrane glycoproteins may also account for the increased agglutination rates with PLL and SBA. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors at the RBC surface are associated with a cytoskeleton within the red cell. Distortion of the RBC cytoskeleton may induce a redistribution of integral membrane proteins and thus modify the surface properties of the cell.

Fatalities in myasthenia gravis; a review of 39 cases with 26 autopsies.

<b>Background:</b> Hypoxemia in community-acquired pneumonia (CAP) has been shown to be a risk factor for death. One of the mechanisms of hypoxemia progression has associated with red blood cells (RBCs) disorders. <b>Aim and objectives:</b> The aim of the present work was to determine metabolic perturbations in RBCs from CAP patients. Control subjects were 32 healthy volunteers without any medication. 34 patients with CAP were on hospitalization and inspection. <b>Methods:</b> The concentration of membrane-bounded hemoglobin was measured in RBCs. Sorption property of RBCs was measured using methylene blue. High erythrocyte membrane permeability was associated with increased methylene blue sorption, which correlated with erythrocyte damage. <b>Results:</b> Our results showed the increase in sorption capacity of RBCs (by 24%, p &lt; 0.05) in comparison with healthy ones. The level of membrane-bounded hemoglobin in RBCs from CAP patients was higher than in control samples (by 47%, p&lt;0.05). <b>Conclusions:</b> Evaluation of sorption capacity of RBCs is considered as indicator of membrane disorders. We surmised that increasing of RBCs sorption property may be connected with low activity NADPH methemoglobin reductase. The increasing membrane-bounded hemoglobin affects RBCs membrane and cytoskeleton proteins disorganization, and effects the changing of RBCs transporting function. Taken together those alterations promote hypoxemia progression.