Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells

The short F-actins in the red blood cell (RBC) membrane skeleton are coated along their lengths by an equimolar combination of two tropomyosin isoforms, Tpm1.9 and Tpm3.1. We hypothesized that tropomyosin’s ability to stabilize F-actin regulates RBC morphology and mechanical properties. To test this, we examined mice with a targeted deletion in alternatively spliced exon 9d of Tpm3 (Tpm3/9d–/–), which leads to absence of Tpm3.1 in RBCs along with a compensatory increase in Tpm1.9 of sufficient magnitude to maintain normal total tropomyosin content. The isoform switch from Tpm1.9/Tpm3.1 to exclusively Tpm1.9 does not affect membrane skeleton composition but causes RBC F-actins to become hyperstable, based on decreased vulnerability to latrunculin-A–induced depolymerization. Unexpectedly, this isoform switch also leads to decreased association of Band 3 and glycophorin A with the membrane skeleton, suggesting that tropomyosin isoforms regulate the strength of F-actin-to-membrane linkages. Tpm3/9d–/–mice display a mild compensated anemia, in which RBCs have spherocytic morphology with increased osmotic fragility, reduced membrane deformability, and increased membrane stability. We conclude that RBC tropomyosin isoforms directly influence RBC physiology by regulating 1) the stability of the short F-actins in the membrane skeleton and 2) the strength of linkages between the membrane skeleton and transmembrane glycoproteins.

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Erythroid and nonerythroid spectrins

Blood ◽

10.1182/blood.v81.12.3173.3173 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3173-3185 ◽

Cited By ~ 113

Author(s):

JC Winkelmann ◽

BG Forget

Keyword(s):

Membrane Skeleton ◽

Structural Knowledge ◽

Direct Relevance ◽

Rbc Membrane ◽

Recent Developments ◽

Alternatively Spliced ◽

Rbc Membrane Skeleton

Abstract Recent developments have contributed important information to understanding the role of spectrins in the RBC membrane skeleton and nonerythroid cells. Many questions can now be framed, informed by structural knowledge of various spectrin subunit types and alternatively spliced variants, that previously could not have been addressed. Their solution in the coming years will likely lead to further advances with direct relevance to biology and medicine.

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Erythroid and nonerythroid spectrins

Blood ◽

10.1182/blood.v81.12.3173.bloodjournal81123173 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3173-3185 ◽

Cited By ~ 2

Author(s):

JC Winkelmann ◽

BG Forget

Keyword(s):

Membrane Skeleton ◽

Structural Knowledge ◽

Direct Relevance ◽

Rbc Membrane ◽

Recent Developments ◽

Alternatively Spliced ◽

Rbc Membrane Skeleton

Recent developments have contributed important information to understanding the role of spectrins in the RBC membrane skeleton and nonerythroid cells. Many questions can now be framed, informed by structural knowledge of various spectrin subunit types and alternatively spliced variants, that previously could not have been addressed. Their solution in the coming years will likely lead to further advances with direct relevance to biology and medicine.

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A novel splicing mutation of the α-spectrin gene in the original hereditary pyropoikilocytosis kindred

Blood ◽

10.1182/blood-2005-05-1813 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4367-4369 ◽

Cited By ~ 13

Author(s):

Daniel B. Costa ◽

Larisa Lozovatsky ◽

Patrick G. Gallagher ◽

Bernard G. Forget

Keyword(s):

Premature Termination Codon ◽

Membrane Skeleton ◽

Molecular Defect ◽

Culture Cells ◽

Rbc Membrane ◽

Tissue Culture Cells ◽

Gene Transcripts ◽

Consensus Splice Site ◽

Defective Allele ◽

Rbc Membrane Skeleton

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia due to abnormalities of the red blood cell (RBC) membrane skeleton. In the original HPP kindred, there is compound heterozygosity for an allele encoding a structural variant of α-spectrin (L207P) and an α-spectrin allele associated with a defect in α-spectrin production. To identify the molecular defect in the production-defective allele, reticulocyte α-spectrin cDNA from one of the original HPP patients was analyzed. Transcripts from the production-defective, non-L207P allele demonstrated a pattern of abnormal splicing between exons 22 and 23, resulting in insertion of intronic fragments with an in-frame premature termination codon. A G to A substitution at position +5 of the donor consensus splice site of IVS 22 was identified in the inserts. Following gene transfer into tissue culture cells, there was complete absence of normally spliced α-spectrin gene transcripts derived from a minigene containing the IVS 22 +5 mutation.

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Targeted Deletion of γ-Adducin in Mice.

Blood ◽

10.1182/blood.v110.11.1723.1723 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1723-1723

Author(s):

Kenneth E. Sahr ◽

Amy J. Lambert ◽

Steven L. Ciciotte ◽

Luanne L. Peters

Keyword(s):

Western Blotting ◽

Peripheral Blood ◽

Osmotic Fragility ◽

Membrane Skeleton ◽

Red Cells ◽

Null Mouse ◽

Exon 2 ◽

Rbc Membrane ◽

Blood Smears ◽

Peripheral Blood Smears

Abstract The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (<5% of normal). These studies show that (1) loss of γ-adducin alone does not significantly impact RBC membrane skeleton structure and function; (2) α- and β-adducin are stable and present at normal levels in the absence of γ-adducin; (3) the loss of γ-adducin in β-adducin null mice does not further exacerbate the β-adducin null HS phenotype; (4) the exacerbated loss of α-adducin in βγ double null RBCs suggests that up-regulated γ-adducin in β-adducin null mice associates in some way with and stabilizes α-adducin in the RBC membrane, but is unable to compensate functionally for the loss of the β subunit. We conclude that the normal function and stable incorporation of adducin into the peripheral membrane skeleton of red cells requires the presence of heterologous αβ binding subunits. Additional studies of adducin null mouse models, including our recently generated α-adducin null strain, will be useful tools in defining adducin functions and interactions in multiple tissues and organs.

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Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries

10.1101/2020.07.11.191494 ◽

2020 ◽

Author(s):

Arman Namvar ◽

Adam J. Blanch ◽

Matthew W. Dixon ◽

Olivia M. S. Carmo ◽

Boyin Liu ◽

...

Keyword(s):

Surface Area ◽

Marked Effect ◽

Volume Ratio ◽

Element Model ◽

Membrane Skeleton ◽

Measurable Effect ◽

Rbc Membrane ◽

Nonlinear Mechanical ◽

Small Channels ◽

Rbc Membrane Skeleton

SummaryThe remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar stiffness. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

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Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of α-Spectrin.

Blood ◽

10.1182/blood.v106.11.1662.1662 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1662-1662

Author(s):

Raymond F. Robledo ◽

Amy J. Lambert ◽

Babette Gwynn ◽

Lucy B. Rowe ◽

Diana M. Gilligan ◽

...

Keyword(s):

Hemolytic Anemia ◽

Membrane Skeleton ◽

Band 3 ◽

Chromosome 1 ◽

Major Protein ◽

Blood Profile ◽

Rbc Membrane ◽

C Terminus ◽

Sds Page ◽

Rbc Membrane Skeleton

Abstract The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

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Comprehensive Analysis of Murine and Human RBC Ghost Proteomes.

Blood ◽

10.1182/blood.v108.11.1569.1569 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1569-1569

Author(s):

Jason M. Wooden ◽

Greg Finney ◽

Michael MacCoss ◽

Diana M. Gilligan

Keyword(s):

Mouse Models ◽

Shotgun Proteomics ◽

Membrane Skeleton ◽

Protein Repair ◽

Knock Out ◽

Human Samples ◽

Rbc Membrane ◽

Northern European ◽

Associated Proteins ◽

Rbc Membrane Skeleton

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Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

Blood ◽

10.1182/blood-2010-02-268458 ◽

2010 ◽

Vol 116 (14) ◽

pp. 2590-2599 ◽

Cited By ~ 59

Author(s):

Jeannette D. Moyer ◽

Roberta B. Nowak ◽

Nancy E. Kim ◽

Sandra K. Larkin ◽

Luanne L. Peters ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Membrane Skeleton ◽

Actin Dynamics ◽

Filament Length ◽

Western Blots ◽

Rbc Membrane ◽

Cellular Dehydration ◽

Rbc Membrane Skeleton

Abstract The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

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Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia.

Blood ◽

10.1182/blood.v110.11.1728.1728 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1728-1728

Author(s):

Jason M. Wooden ◽

Greg L. Finney ◽

Michael J. MacCoss ◽

Luanne L. Peters ◽

Diana M. Gilligan

Keyword(s):

Mouse Models ◽

Hemolytic Anemia ◽

Membrane Skeleton ◽

Tandem Mass ◽

Secondary Effects ◽

Knock Out ◽

Rbc Membrane ◽

Protein 4.2 ◽

Knock Out Mice ◽

Rbc Membrane Skeleton

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up’ analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.

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Quantitative Mass Spec Analysis Of RBCs From Patients With Hereditary Spherocytosis

Blood ◽

10.1182/blood.v122.21.4659.4659 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4659-4659

Author(s):

Bhuvaneswari Ramkumar ◽

David Kakhniashvili ◽

Steven Goodman ◽

Diana Gilligan

Keyword(s):

Mass Spectrometry ◽

Hereditary Spherocytosis ◽

Protein Composition ◽

Membrane Skeleton ◽

Mass Spectrometry Analysis ◽

Itraq Labeling ◽

Rbc Membrane ◽

Mass Spec ◽

Sds Page ◽

Rbc Membrane Skeleton

Introduction Spherocytic RBCs have increased fragility due to abnormalities of the components of the RBC membrane skeleton. From various studies about 30-40% of patients showed no molecular defect using standard Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Mass spectrometry analysis (proteomics) would provide a deeper insight into changes in RBC membrane skeleton protein composition in patients with hereditary spherocytosis (HS). The objective of this study is to identify biomarkers that explain phenotypic heterogeneity and severity of hemolysis and variability within the same family using proteomic tools. Methods Blood samples from affected and unaffected family members of patients with hereditary spherocytosis at Upstate Medical University have been collected after obtaining informed consent. The family presented here includes the affected mother (OS2) and affected grandfather (OS3) of the female patient (OS1) with HS. The grandfather underwent splenectomy many years ago. Samples were collected from the three affected members and the unaffected grandmother (OS4). Complete blood count, peripheral smears, reticulocyte percentage (%) and absolute reticulocyte count were performed on those samples. RBC ghosts were prepared by osmotic lysis and were fractionated into membrane and cytosol. Membrane fractions were analyzed by SDS-PAGE. Membrane and cytosol fractions are being analyzed by 4plex Isobaric tags for relative and absolute quantitation (iTRAQ) labeling. The four individual samples are labeled with amine-modifying four different iTRAQ reagents and then combined. The labeled samples are separated by SDS-PAGE, and then regions of the gel are cut out and subjected to in-gel trypsin digest. The labeled peptides are then analyzed by tandem mass spectrometry and relative abundance can be assigned to each of the four labeled samples within the same analysis, corresponding to the three patients and one control. This approach minimizes any differences in sample processing because all four samples are analyzed simultaneously on the mass spectrometer. Preliminary Results Reticulocyte % and absolute retic count were highest for our patient (13.5 %) compared to her mother (9.93%) and her grandfather (3.34%) who was post splenectomy. Hemoglobin and hematocrit were also lower for our patient (12.8/36.0) compared to her mother (13.3/37.1) and grandfather (16.9/48.1). Coomassie blue SDS-PAGE of RBC ghosts did not show any differences in protein composition at this level of detection. Mass spec analysis using 4plex iTRAQ labeling is currently underway. Conclusions Patients with hereditary spherocytosis exhibit a wide range of phenotypes even within the same family. Protein differences are often not detected at the level of SDS-PAGE. In-depth analysis by quantitative mass spectrometry allows the identification of differences that can correlate to severity of illness and aid in choices regarding treatment modalities. Disclosures: No relevant conflicts of interest to declare.

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