Red Blood Cell Cytoskeleton Research Articles

Effective cell membrane tension protects red blood cells against malaria invasion.

A critical step in how malaria parasites invade red blood cells (RBCs) is the wrapping of the membrane around the egg-shaped merozoites. Recent experiments have revealed that RBCs can be protected from malaria invasion by high membrane tension. While cellular and biochemical aspects of parasite actomyosin motor forces during the malaria invasion have been well studied, the important role of the biophysical forces induced by the RBC membrane-cytoskeleton composite has not yet been fully understood. In this study, we use a theoretical model for lipid bilayer mechanics, cytoskeleton deformation, and membrane-merozoite interactions to systematically investigate the influence of effective RBC membrane tension, which includes contributions from the lipid bilayer tension, spontaneous tension, interfacial tension, and the resistance of cytoskeleton against shear deformation on the progression of membrane wrapping during the process of malaria invasion. Our model reveals that this effective membrane tension creates a wrapping energy barrier for a complete merozoite entry. We calculate the tension threshold required to impede the malaria invasion. We find that the tension threshold is a nonmonotonic function of spontaneous tension and undergoes a sharp transition from large to small values as the magnitude of interfacial tension increases. We also predict that the physical properties of the RBC cytoskeleton layer-particularly the resting length of the cytoskeleton-play key roles in specifying the degree of the membrane wrapping. We also found that the shear energy of cytoskeleton deformation diverges at the full wrapping state, suggesting the local disassembly of the cytoskeleton is required to complete the merozoite entry. Additionally, using our theoretical framework, we predict the landscape of myosin-mediated forces and the physical properties of the RBC membrane in regulating successful malaria invasion. Our findings on the crucial role of RBC membrane tension in inhibiting malaria invasion can have implications for developing novel antimalarial therapeutic or vaccine-based strategies.

RBC eNOS Transduces Regional O2 Gradients to Orchestrate RBC O2 Delivery Phenotype during Circulatory Transit

BACKGROUND: The role of endogenous endothelial NO synthase (eNOS) in mature RBCs is poorly understood. We have previously demonstrated functionally O2-dependent eNOS migration in RBCs, mediating (via s-nitrosylation) glycolytic metabolon assembly/disassembly during circulation in support of antioxidant systems during stress. We have further explored eNOS migration and now propose the following mechanism along with its functional significance. We (and others) suggest that hemoglobin (Hb) conformational change resulting from deoxygenation leads to its docking at the cytoplasmic domain of Band 3 (cdB3). We propose that (1) cdB3 consequently undergoes a conformational change that is transduced by Piezo1 (a mechanosensitive ion channel located in the RBC membrane); (2) Piezo1 activation results in calcium entry into the RBC, which (3) aids in the disassembly of the RBC cytoskeleton, (4) facilitates eNOS migration to the membrane (where calmodulin is mainly localized - thereby bringing these two important proteins together), and (5) binds to calmodulin, activating eNOS to generate nitric oxide (NO) - which then influences RBC phenotype that facilitates O 2 delivery, specifically related to RBC deformability and regulation of Hb~O 2 release, via enhanced Bohr effect. METHODS: Heparinized human, eNOS (-/-), or wt murine (C57BL/6J) red blood cells (RBCs) were imaged (Nikon) for the analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the Piezo1 agonist Yoda1, or a membrane tension buffer grammostola mechanotoxin #4 (GSMTx4; decoupling Piezo1), or carbon monoxide (CO) to maintain R-state Hb conformation upon RBC deoxygenation. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, protein 4.1, or eNOS), then with the secondary antibody prior to visualization. Interaction between eNOS and cytoskeleton proteins, as well as calmodulin, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader, and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. To assess the functional significance of this mechanism, Hb-O 2 affinity was determined from murine blood (eNOS (-/-) or wt). Oxygen dissociation curves (ODCs), were constructed at 2 fixed CO 2 levels (thin film rotating tonometer; in conjunction with a gas blender; with samples measured by arterial blood gas machine).Bohr effect was assessed by determining the area between the ODCs (ABC) at the 2 fixed CO2 levels (~40mmHg and ~70mmHg). RESULTS: We observed several significant findings related to red RBC deoxygenation. Upon RBC deoxygenation, there is an increase in calcium levels indicated by the Fluo3 signal, which is Piezo1 dependent. Cytoskeleton proteins, such as Actin, Spectrin, and protein 4.1, undergo disassembly upon RBC deoxygenation. This disassembly process is contingent on both Piezo1-mediated calcium flux and the conformational changes in hemoglobin (Hb) (and not pO 2). We found that eNOS is associated with 4.1 throughout the oxy/deoxy cycle and that eNOS exhibits the same migratory pattern as cytoskeleton proteins to the RBC membrane, where calmodulin is located, irrespective of the RBC's oxygenation state. Additionally, when eNOS localizes in the membrane, it exhibits interaction with calmodulin. The generation of nitric oxide (NO) by eNOS, as indicated by the DAF-FM signal, increases in a manner dependent on both calcium, Piezo1 and Hb conformational transition. The functional consequence of this mechanism appears to be related to O 2 delivery phenotype during circulatory transit, with cell deformability being affected in addition to an inhibition of the Bohr effect in eNOS (-/-) compared to wt mice (2006 ± 61 vs 1892 ± 69 ABC in WT (n = 3) vs eNOS (-/-) (n = 4), respectively; p = 0.0747). SUMMARY: Upon circulatory transit, RBC deoxygenation triggers the activation of eNOS. This activated eNOS is associated with Piezo1-mediated calcium flux, which is facilitated by calmodulin. These events are coordinated with the migration of eNOS within the red blood cell between the cytosol and the cell membrane. The migration of eNOS is influenced by cytoskeleton proteins.

A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections.

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.

Nanoscale Changes on RBC Membrane Induced by Storage and Ionizing Radiation: A Mini-Review.

The storage lesions and the irradiation of blood cellular components for medical procedures in blood banks are events that may induce nanochanges in the membrane of red blood cells (RBCs). Alterations, such as the formation of pores and vesicles, reduce flexibility and compromise the overall erythrocyte integrity. This review discusses the alterations on erythrocytic lipid membrane bilayer through their characterization by confocal scanning microscopy, Raman, scanning electron microscopy, and atomic force microscopy techniques. The interrelated experimental results may address and shed light on the correlation of biomechanical and biochemical transformations induced in the membrane and cytoskeleton of stored and gamma-irradiated RBC. To highlight the main advantages of combining these experimental techniques simultaneously or sequentially, we discuss how those outcomes observed at micro- and nanoscale cell levels are useful as biomarkers of cell aging and storage damage.

Red Blood Cell (RBC) Echinocyte SEM Structure Directly Affected by Oxygen, Heat and F‐actin Destabilization

The RBC cytoskeleton is a spectrin-actin meshwork tethered to the integral membrane protein, band 3, through several proteins, including ankyrin. Their interactions affect membrane curvature, mechanics, microdomain formation, and may participate in oxygenation of hemoglobin (Hb) as deoxyHb displaces ankyrin to bind band 3. Our focus is to understand how hypoxia and elevated temperature cause RBC shape changes that may impact microvascular rouleaux formation and vessel blockage with thermal burn injury. We hypothesized that both oxygen and F-actin destabilization are required for shape change. Washed human RBCs (BioIVT) suspended in saline with 1% DMSO, 3.7μM Jasplakinolide (JASPA, polymerizes F-actin), 26.14μM Latrunculin A (LatA, depolymerizes F-actin), 0.5μM Cytochalasin D (CytoD, disrupts cytoskeleton), 2μM 4,4ʹ-diisothiocyano-2,2ʹ-stilbenedisulfonic acid (DIDS, binds band 3, untethers cytoskeleton from membrane) or control (platelet poor plasma, PPP). Each was tested at 2 temperatures (T: 37, 45°C) and 3 oxygen levels (O2: 0, 5, 10%). At T/O2, suspensions were fixed with 1.5% paraformaldehyde 1.5% glutaraldehyde, then with OsO4. Samples were serially dehydrated in ethanol, and coated with 12-15nm Au-Pd in Ar (6mA, Polaron E5100). SEM images (FEI Nova NanoSEM 450, high vacuum mode 4-5 mm working distance, beam 2kV, Everhart-Thornley Detector) were obtained and analyzed using ImageJ (NIH) for 44 samples and 656 RBC. RBC long and short diameters were obtained. For echinocytes (ECH), ‘edge’ spicule (seen on periphery), height, H, width at base (BW) and mid-height (MW) were measured; ‘face’ spicule (seen on presenting face) topology was measured (inter-node distances, nearest neighbors, angles). RBC were classified per the clinical standard as biconcave (discocyte), ECH-I (short broad spicules, flat cell), ECH-II (formed spicules, elliptical cell), ECH-III (well formed spicules, spherical). Independent of treatment, T or O2, we first confirmed that spicule shape progressed to taller with a narrower base, with closer packed conformation as ECH progressed from I to III. Independent of treatment or O2, spicules at 45°C for ECH-I were smaller in H and BW/MW, and more closely packed than at 37°C; ECH-II/III only displayed closer packing. In PPP ECH-I spicules at 0%/37°C were broader and further spaced, while spicules at 10%/45°C were significantly smaller in BW/MW and closer packed; ECH-II/III were not affected by oxygen. Concentrating on ECH-I, DIDS removed the effects on W and spacing at all oxygen/temps. Actin destabilization (CytoD) removed the effect of O2 and T on W, but not spacing; while depolymerization (LatA) had little effect on W or spacing. Actin polymerization (JASPA) had no effect on W, but removed the effect of O2 on spacing. Thus, decoupling the cytoskeleton from band 3 (0% oxygen or DIDS) resulted in fewer broad spicules; depolymerization only (LatA) had little effect, while destabilization (CytoD) reduced the W but not spacing of spicules, and polymerization (JASPA) affected spacing but not W.

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Improving the EMA Binding Test by Using Commercially Available Fluorescent Beads.

Hereditary spherocytosis (HS) is a common anemia caused by germline mutations in red blood cell cytoskeleton proteins. The flow cytometry-based eosin-5′-maleimide (EMA) binding test is most frequently employed for reliable diagnostics. To perform this test, a number of healthy and ideally also age-matched controls are required, which can be challenging and complicates interlaboratory comparisons. To overcome this limitation, we modified the EMA binding test by replacing healthy controls with commercially available fluorescent beads. Blood samples from 289 individuals with suspected HS were analyzed using the EMA binding test with fluorescent beads and benchmarked against regular EMA binding test using two control samples. Using osmotic gradient ektacytometry as validation, 112 individuals (38.8%) were diagnosed with HS. Performance of the modified EMA binding test was not compromised (accuracy 90.3%) compared to EMA binding test using matched controls (accuracy 88.6%). Based on these findings, we conclude that the modified EMA binding test with fluorescent beads is an attractive alternative, especially in laboratories without easy access to matched controls. Furthermore, as fluorescent beads are stable and easily commutable, they could facilitate both interlaboratory comparisons and quality assessment programs.

Actin Destabilization Enhances Thermally‐induced Morphological Abnormalities in Erythrocytes

Thermal burns are a significant clinical problem with limited treatment options. Burn area progresses radially over several days, blocking oxygenated flow to a progressively increasing region. By 4 hours post‐burn, red blood cell (RBC) aggregates form at the burn margin, without evidence of fibrin clots, indicating a potential for early intervention (Clark, 2013). The present study focuses on understanding the early aggregation process. The RBC’s biconcave shape is due to the cytoskeletal structure that is tethered to the membrane; many pathologies occur when this is disrupted. The RBC cytoskeleton includes spectrin‐actin meshwork that affects membrane curvature, mechanics, and microdomain formation. Our specific hypothesis is that elevated temperatures in arterial oxygen conditions (10%) contributes to the actin cytoskeleton destabilization, thus permitting membrane deformation. Washed human RBCs (BioIVT, Westbury, NY) were suspended in saline (PBS) containing 1% DMSO, 3.7μM Jasplakinolide (JASPA, polymerizes F‐actin), 26.14μM Latrunculin A (LatA, depolymerizes F‐actin), 0.5μM Cytochalasin D (CytoD, disrupts all cytoskeletal integrity), 2μM 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS, binds to band 3, untethering the cytoskeleton from the membrane) (1% DMSO) or control (PBS and platelet poor plasma, PPP). All conditions were exposed to five temperatures (37, 41, 43, 45, or 49°C) and three oxygen levels (0, 5, or 10% O2) for 10 minutes. Suspensions were fixed with 4% paraformaldehyde (PFA) at the test temperature/oxygen and imaged using a 60X water immersion objective. Singlet RBCs were scored by their morphology, in accordance with clinical standards, and identified by degree of echinocyte (I, II, and III), type of non‐echinocyte abnormal shape, or as discocytes. With increasing temperature, the population shifted from the discocyte shape for all control conditions. Increasing oxygen (in plasma) likewise shifted RBC morphology to pathological shapes, but with a less pronounced effect. CytoD, DIDS, JASPA and LatA each eliminated the discocyte populations at all temperatures and oxygen levels. Cells were also prepared for SEM imaging using 1.5% glutaraldehyde and 1.5% PFA and serial dehydration. RBC shapes obtained from brightfield images (current clinical practice) were compared to shapes obtained from SEM images. Population distribution of shapes from these two image pools were not different. Overall, we show that heat and increasing oxygen promotes pathological RBC morphologies. Deliberate disruption of the actin cytoskeleton induces these morphologies, which is exacerbated by increasing temperature. Further, LatA and JASPA cause similar population distributions of shapes, thereby indicating any imbalance of the equilibrium between filamentous and globular actin triggers morphological abnormalities.

Improvement in Red Blood Cell Physiology in Children with Sickle Cell Anemia Receiving Voxelotor

Background: Sickle hemoglobin (HbS) under conditions of deoxygenation polymerizes to cause sickling of red blood cells (RBCs) and other rheological abnormalities. Voxelotor has been previously shown in a preclinical model of sickle cell disease (SCD) to increase HbS affinity to oxygen, thus reducing its polymerization and sickling with subsequent increase in the half-life of RBCs. We hypothesized that given this mechanism of action, we would observe improvements in RBC physiology in patients receiving voxelotor. In the Phase 3 GBT HOPE trial, the use of voxelotor in patients with SCD caused a significant reduction in markers of hemolysis and anemia. Ektacytometry is considered the gold standard to study deformability of RBCs with membrane protein disorders. The deformability of RBCs can be assessed using a defined value of shear stress with an increasing osmotic gradient (osmoscan) as well as with a newer technology to subject these cells to gradual deoxygenation (oxygenscan). Both assays can be measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, NL). In this pilot study, we analyzed samples from patients with SCD receiving voxelotor, before and 12 weeks after starting therapy to assess the benefits of voxelotor on RBC physiology. Methods: Our pilot study obtained whole blood from children ages 4-11 years with SCD, who were enrolled in the IRB approved GBT 440-007 clinical trial (NCT02850406; a study evaluating multiple doses of voxelotor at 1500 mg/day equivalent exposure to adults based on body weight) at Emory University/Children's Healthcare of Atlanta. All participants in this cohort continued their stable, optimal hydroxyurea dose during treatment with voxelotor. The below measurements were performed on the pre-dose and Week 12 visit samples. Deformability of RBCs was performed at a shear stress of 30 Pa and varying osmolality gradients (0-600 mOsm/Kg) for Osmoscan. Omin corresponds to the value of the hypotonic osmolality, where 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface area:volume ratio. Maximal deformability or Elongation Index (EImax) near isotonic osmolality informs us of the RBC cytoskeleton mechanics and Ohyper, the osmolality corresponding to 50% of the Elmax, provides information regarding the cytoplasmic viscosity. Oxygenscan was performed but under controlled deoxygenation using nitrogen. Point of Sickling (POS) is a point on the curve during deoxygenation when sickling begins, and EImin corresponds when sickle RBCs can least elongate. Oxygen dissociation curves were obtained using a HemOx Analyzer (TCS Scientific). Complete blood count parameters were determined on a clinical laboratory hematology analyzer (ADVIA, Siemens). Data was analyzed with Prism using a paired T-test. Results: Both pre-dose and Week 12 visit samples were available for 10 participants. Mean hemoglobin at baseline was 9.0 g/dL (7.6-10.0) and at 12 weeks, 10.3 g/dL (8.2-12.3). Six out of 10 participants had a hemoglobin response at Week 12 (defined as an increase in Hb from baseline by >1 g/dL), of which 5 had hemoglobin over 10 g/dL. Mean % change in percentage of reticulocytes was -17.0%. Significant improvement in EImax on osmoscan was noted at Week 12 (p=0.0147), suggesting RBCs were more deformable with improved cytoskeleton mechanics. In addition, oxygenscan curves shifted upwards towards normal with a significant increase in EImax (p=0.0347) and EImin (p=0.0079). These findings combined with a decrease in POS (p=0.0001) during deoxygenation suggests that at low oxygen tension, voxelotor treated RBCs were more deformable possibly from reduced HbS polymer inside these cells. Significant reductions in P50 (p=0.0011) and P20 (p=0.0001) with a left shift of the oxygen dissociation curve further demonstrates the effect of voxelotor on RBCs. Discussion: Voxelotor therapy in children with HbSS is associated with reductions in anemia and reticulocyte response, and recovery in RBC health as early as 12 weeks of treatment. Voxelotor's ability to inhibit HbS polymerization and RBC sickling is associated with specific modulation in red cell rheology at normoxic and deoxygenating conditions. Left shifted oxygen dissociation curves confirm voxelotor's ability to increase oxygen affinity. These findings suggest that voxelotor improves RBC deformability and anemia and delays the initiation of RBC sickling. Figure Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Baratz:Prolong Pharmacuticals: Honoraria; Global Blood Therapeutics: Research Funding. Pochron:Global Blood Therapeutics: Employment, Equity Ownership. Dixon:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapeutics: Employment, Equity Ownership. Lehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Brown:Pfizer: Research Funding; Novartis, Inc: Research Funding; Imara, Inc: Consultancy, Research Funding; Global Blood Therapeutics, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Archer:AstraZeneca: Research Funding; Prolong Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding.

The Spectrum of SPTA1-Associated Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is the most common red blood cell (RBC) membrane disorder causing hereditary hemolytic anemia. Patients with HS have defects in the genes coding for ankyrin (ANK1), band 3 (SLC4A1), protein 4.2 (EPB42), and α (SPTA1) or β-spectrin (SPTB). Severe recessive HS is most commonly due to biallelic SPTA1 mutations. α-spectrin is produced in excess in normal erythroid cells, therefore SPTA1-associated HS ensues with mutations causing significant decrease of normal protein expression from both alleles. In this study, we systematically compared genetic, rheological, and protein expression data to the varying clinical presentation in eleven patients with SPTA1-associated HS. The phenotype of HS in this group of patients ranged from moderately severe to severe transfusion-dependent anemia and up to hydrops fetalis which is typically fatal if transfusions are not initiated before term delivery. The pathogenicity of the mutations could be corroborated by reduced SPTA1 mRNA expression in the patients’ reticulocytes. The disease severity correlated to the level of α-spectrin protein in their RBC cytoskeleton but was also affected by other factors. Patients carrying the low expression αLEPRA allele in trans to a null SPTA1 mutation were not all transfusion dependent and their anemia improved or resolved with partial or total splenectomy, respectively. In contrast, patients with near-complete or complete α-spectrin deficiency have a history of having been salvaged from fatal hydrops fetalis, either because they were born prematurely and started transfusions early or because they had intrauterine transfusions. They have suboptimal reticulocytosis or reticulocytopenia and remain transfusion dependent even after splenectomy; these patients require either lifetime transfusions and iron chelation or stem cell transplant. Comprehensive genetic and phenotypic evaluation is critical to provide accurate diagnosis in patients with SPTA1-associated HS and guide toward appropriate management.

S902 THE COMPLEXITY OF GENOTYPE‐PHENOTYPE CORRELATIONS IN HEREDITARY SPHEROCYTOSIS: A COHORT OF 95 PATIENTS

Background:Hereditary spherocytosis (HS) presents as a highly heterogeneous disease, both phenotypically and genetically. Disease severity is directly related to the extent of loss of surface area of the red cell. The diagnosis is multifaceted and includes functional testing of red cell deformability. Until recently, establishing or confirming the diagnosis at the DNA level was only rarely done. With increased use of Next‐Generation‐Sequencing (NGS) techniques in diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. However, insight into the impact of genetic defects on the function of membrane proteins, and consequently on phenotypic presentation, is still limited.Aims:Explore genotype‐phenotype correlation in a large cohort of patients with HS.Methods:Retrospective analysis on a cohort of 95 HS patients genotyped by targeted‐NGS (SPTA1, SPTB, ANK1, SLC4A1, EPB41, EPB42, RHAG) as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands).Results:Our cohort included 95 patients suspected to have HS. In 85/95 (89%) of the patients a pathogenic mutation was identified, including 56 novel mutations. Mutations in SPTA1 were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 mutations showed autosomal dominant inheritance. ANK1 mutations accounted for 27% (23/85 patients) and SPTB mutations for 20% (17/85 patients) of the cases, all showing autosomal dominant inheritance. Moderate or severe HS was more frequently observed in patients with SPTB or ANK1 mutations, reflected by significantly lower hemoglobin concentrations (SPTB 10.5 g/dL, ANK1 10.8 g/dL compared to SLC4A1 13.2 g/dL, both p < 0.05) and higher absolute reticulocyte counts (SPTB 580x10^9/L, ANK1 506x10^9/L compared to SPTA1 298x10^9/L and SLC4A1 259x10^9/L, all p < 0.01) in non‐splenectomized patients.We investigated the correlation between phenotype and genetic mutations using protein structure information (Figure). Mutations positioned in the spectrin‐association domains of ANK1, SPTA1 and SPTB resulted in a more severe phenotype. Interestingly, deletion of the whole SPTB gene resulted in a mild phenotype, while the majority of other pathogenic mutations in SPTB were associated with a more severe disease phenotype, suggesting that incorporation of truncated or dysfunctional protein occurs, which might be more harmful than an absolute decrease of otherwise normal protein.Our cohort included nine families and broad phenotypic heterogeneity was observed in four of them. Phenotypic heterogeneity could not be directly explained by the underlying genotype, suggesting that other (genetic) yet‐unknown modifiers contribute to disrupted interactions in the red cell cytoskeleton. To identify alternative predictors of clinical severity we analyzed red cell Osmoscan profiles from the Laser‐assisted Optical Rotational Cell Analyzer. Data were available in a subset of our cohort (44 not splenectomized patients). We observed a clear association between clinical disease severity and the maximal deformability, and the area under the curve (respectively r = −0.48, p < 0.01, and r = −0.38, p = 0.01).Summary/Conclusion:Genotype‐phenotype prediction in HS requires additional insight in the consequences of pathogenic mutations for assembly of and dynamic interactions in the red blood cell cytoskeleton. Currently, in conjunction to the molecular deficit, functional analysis of red cell deformability adds to the prediction of phenotypic variability.image

PS1197 ASSESSING THE EMA BINDING TEST AND OSMOTIC GRADIENT EKTACYTOMETRY IN THE DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS DEFINED BY THE PRESENCE OF UNDERLYING DIAGNOSTIC MUTATIONS

Background:Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin‐5’‐maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next‐generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear.Aims:To improve accuracy, robustness, and cost‐effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS.Methods:We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA‐binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser‐assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α‐spectrin, β‐spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations.Results:A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations.The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry‐derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry‐derived parameter EImax (<0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin >166 mOsm/kg and EImax <0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty‐seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values <40 had mutations only in the SPTA1 gene.Summary/Conclusion:Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and <0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.image

PS1198 MOLECULAR DIAGNOSIS OF RARE CONGENITAL ANEMIAS USING TARGETED NEXT-GENERATION SEQUENCING IN INDIAN PATIENTS

Background: Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin-5’-maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next-generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear. Aims: To improve accuracy, robustness, and cost-effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS. Methods: We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA-binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α-spectrin, β-spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations. Results: A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations. The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry-derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry-derived parameter EImax (<0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin >166 mOsm/kg and EImax <0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty-seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values <40 had mutations only in the SPTA1 gene.Summary/Conclusion: Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and <0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.

The Plasmodium falciparum MESA erythrocyte cytoskeleton-binding (MEC) motif binds to erythrocyte ankyrin

The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5C domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif’s primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton.

We present Skan (Skeleton analysis), a Python library for the analysis of the skeleton structures of objects. It was inspired by the “analyse skeletons” plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API) allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan’s measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM)-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions.

The role of the red blood cell in host defence against falciparum malaria: an expanding repertoire of evolutionary alterations.

The malaria parasite has co-evolved with its human host as each organism struggles for resources and survival. The scars of this war are carried in the human genome in the form of polymorphisms that confer innate resistance to malaria. Clinical, epidemiological and genome-wide association studies have identified multiple polymorphisms in red blood cell (RBC) proteins that attenuate malaria pathogenesis. These include well-known polymorphisms in haemoglobin, intracellular enzymes, RBC channels, RBC surface markers, and proteins impacting the RBC cytoskeleton and RBC morphology. A better understanding of how changes in RBC physiology impact malaria pathogenesis may uncover new strategies to combat the disease.

High Aspect Ratio Sub-Micrometer Channels Using Wet Etching: Application to the Dynamics of Red Blood Cell Transiting through Biomimetic Splenic Slits.

Nanoparticles delivering drugs, disseminating cancer cells, and red blood cells (RBCs) during splenic filtration must deform and pass through the sub-micrometer and high aspect ratio interstices between the endothelial cells lining blood vessels. The dynamics of passage of particles/cells through these slit-like interstices remain poorly understood because the in vitro reproduction of slits with physiological dimensions in devices compatible with optical microscopy observations requires expensive technologies. Here, novel microfluidic PDMS devices containing high aspect ratio slits with sub-micrometer width are molded on silicon masters using a simple, inexpensive, and highly flexible method combining standard UV lithography and anisotropic wet etching. These devices enabled revealing novel modes of deformations of healthy and diseased RBCs squeezing through splenic-like slits (0.6-2 × 5-10 × 1.6-11 µm3 ) under physiological interstitial pressures. At the slit exit, the cytoskeleton of spherocytic RBCs seemed to be detached from the lipid membrane whereas RBCs from healthy donors and patients with sickle cell disease exhibited peculiar tips at their front. These tips disappeared much slower in patients' cells, allowing estimating a threefold increase in RBC cytoplasmic viscosity in sickle cell disease. Measurements of time and rate of RBC sequestration in the slits allowed quantifying the massive trapping of spherocytic RBCs.

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4.

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase-like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.

Wnt Signaling in Red Blood Cells

Introduction: Red blood cells (RBCs) are the most common cell type in the human body.These cells deliver oxygen to the body's tissues and are composed of a cytoplasm that is rich in hemoglobin and is surrounded by a membrane that is essential for the cells function by providing properties such as stability and deformability. The membrane is composed of a lipid bilayer, transmembrane proteins, and a filamentous meshwork of proteins such as actin and adducin that forms the cells cytoskeleton along the entire cytoplasmic surface of the membrane. RBCs lack a nucleus and other cellular organelles that enable gene expression. Thus, it has been assumed, that signaling cascades such as the Wnt signaling pathway are not active in RBCs.The Wnt pathways are fundamental for normal development and homeostasis and regulate, among other, cell growth, motility and differentiation. Although there is no functional data connecting between Wnt signaling and RBCs, previous proteomic studies have shown that some of the non-canonical Wnt signaling components, such as specific small GTPases and kinases are present in RBCs. In this study, we show for the first time that Wnt ligands activate signaling cascades in RBCs.Methods: RBCs were collect from healthy donors and treated with different Wnt ligands. A large number of methods were used to evaluate the RBCs morphological properties, life span, vitality, flexibility and protein expression patterns.Results: Our results clearly show that different Wnt ligands can dramatically increase the live span of RBCs by affecting the cells membrane cytoskeleton. Some of the Wnts affects include activation of GTPases such as Rac, JNK and RhoA, which lead to actin modification. These changes in the actin cytoskeleton result in increased membrane stability. In parallel, treating RBCs with Wnts leads to activation of PKC and RhoA resulting in phosphorylation of adducin which in turn increases the cells membrane flexibility and improves the vitality of the cell.Summary: Our novel findings indicate that the non-canonical Wnt signaling pathway in RBCs is active and can stabilize the RBCs cytoskeleton enhancing the vitality, deformability and life span of the cell.These novel findings may help in the development of new therapeutic strategies for people suffering from different hemolytic disease that effect the RBCs cytoskeleton and provide new insights into the improvement of stored RBC units. DisclosuresNo relevant conflicts of interest to declare.