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PubMed1982

Proteins involved in the attachment of actin to the plasma membrane.

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes alpha-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle alpha-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in cross-reacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.

Actinin↗
PubMed1999

Red blood cell membrane disorders.

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

Blood Platelet Disorders↗
PubMed1989

Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives.

The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.

Aldehydes↗
PubMed2004

The irreversibly sickled cell: a perspective.

The irreversibly sickled cell (ISC) is poorly deformable, dehydrated, of short life span, correlated to hemolysis, and a contributor to the pathophysiology of vaso-occlusive (VOC) episodes. The altered redox status and increased oxygen radical levels within high density sickle cells leads to oxidative damage and glutathiolation of cysteine residues. The formation of a disulfide bridge between Cys 284 and Cys 373 in ISC beta-actin leads to actin filaments which depolymerize poorly at 37 degrees C. Glutathiolation of cysteines within spectrin results in this key membrane skeletal protein losing it's E2/E3 ubiquitin-ligating/conjugating activity and therefore ability to self ubiquitinate. The resulting loss of ubiquitination in ISC alpha-spectrin repeats 20/21 causes a higher affinity ISC spectrin-4.1-actin ternary complex. Therefore, reversible oxidative damage to beta-actin and loss of ubiquitination of alpha-spectrin leads to an ISC membrane skeleton that disassembles poorly at 37 degrees C. The result is a membrane skeleton which is "locked" because it cannot disassemble or reassemble. N-acetylcysteine (NAC) is an antioxidant which raises intracellular reduced glutathione levels, and blocks the formation of ISCs in vitro. NAC, in a phase II human trial, caused a downward trend in ISCs, significantly decreased dense cells, and substantially decreased the rate of VOC episodes.

Acetylcysteine↗
PubMed1992

Oxidative erythrocyte membrane damage in hereditary spherocytosis.

The occurrence, in Hereditary Spherocytosis, of an oxidative damage to red blood cell membranes was studied by "in vitro" treatment of the erythrocytes with tert-butylhydroperoxide, methylene blue, or phenylhydrazine. Spherocytes were found to be more sensitive than normal erythrocytes to the action of these drugs. Tert-butylhydroperoxide caused a more intense lipid peroxidation as well as more extensive membrane protein alterations, namely spectrin degradation, formation of high molecular weight aggregates, and globin binding to the membrane. Marked spectrin degradation was also induced by methylene blue and by phenylhydrazine, which differed from each other for their effects on the generation of membrane-bound globin and of intermediate proteolysis products. Spectrin appeared therefore to be, in Hereditary Spherocytosis, a highly sensitive target to oxidative stress, a phenomenon which may, also "in vivo", increase the rate of spectrin loss thus enhancing erythrocyte fragility.

Blood Proteins↗
PubMed1990

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes.

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Hereditary spherocytosis and related disorders.

A number of abnormalities in cellular physiology have been observed in hereditary spherocytes, including alterations in shape, membrane cation permeability and deformability, intracellular metabolism and tendency for splenic entrapment. Many observations have been observed only in a subset of patients with HS and may studies have not been confirmed. Therefore, it is likely that there is heterogeneity with regard to the specific molecular cause of the disease. The major research problem has been to determine primary molecular defects in HS. Much evidence supports a molecular defect in the erythrocyte membrane skeleton and three abnormalities involving spectrin have been demonstrated to be directly related to HS. First, spectrin deficiency has been shown in autosomal recessive spherocytosis in mouse mutants and partial deficiency observed in all human patients with HS. Second, a specific functional defect in spectrin purified from the red cells of some kindreds with autosomal dominant HS has been identified: lack of binding capacity for protein 4.1. Third, a less well characterized functional abnormality has been described in which spectrin binds more tightly to the erythrocyte membrane. These defects may, by an unidentified mechanism, contribute to the spheroidal shape and haemolytic disease ameliorated by splenectomy. More definitive studies are necessary in order to determine the origins of HS. Such studies require: Use of appropriate controls for splenectomy and young red cell age, Tracing a defect through affected family members, Verifying that a defect corresponds to the appropriate heredity pattern, for example that a heterozygote for an autosomal dominant defect had 50% abnormal protein, Differentiating the effects of splenic or circulatory conditioning from the primary red cell defects, Verifying that the defect is present in the intact cell and is not secondary to experimental manipulations, Distinguishing an unrelated, linked polymorphism from the primary mutation responsible for the disorder. Finally, the pathophysiology of the disease will have to be explained on the basis of the primary molecular defect, as well as the mechanism of all secondary physiological changes in the hereditary spherocyte.

Erythrocyte Membrane↗
PubMed1983

Phosphoprotein phosphatases in human erythrocyte cytosol.

More than 97% of spectrin phosphatase activity in human erythrocyte hemolysate was recovered in cytosol. The cytosolic phosphatase activity was resolved into four peaks, namely phosphatases I (22%, Mr = 180,000), II (3%, Mr = 42,000), III (8%, Mr = 177,000), and IV (62%, Mr = 104,000), by aminohexyl-Sepharose column chromatography. Although these phosphoprotein phosphatases also catalyzed the dephosphorylation of phosphorylase a, glycogen synthase b, and phosphorylated H1 and H2B histones, the phosphatases differed from each other in preferences for substrates and the Mg2+ or Mn2+ requirements for their activities. The treatment with 80% ethanol converted phosphatases I, III, and IV to Mr = 31,000 forms which had essentially the same physical and catalytic properties. By contrast, the molecular weight and catalytic properties of phosphatase II, which was Mg2+- or Mn2+-dependent, were not changed by the same ethanol treatment. The major spectrin phosphatase, phosphatase IV, was purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-gel electrophoresis revealed that the enzyme was composed of one 32,000-Da polypeptide (alpha) and one 69,000-Da polypeptide (beta). Km values of the enzyme for phosphorylated spectrin and H2B histone were 1.63 +/- 0.45 and 48.2 +/- 7.6 microM, respectively. The spectrin phosphatase activity was stimulated about 2-fold by 5-25 mM Mg2+, but was completely inhibited by the same concentration of Mn2+. Physiological concentrations of adenine nucleotides, 2,3-diphosphoglyceric acid, cyclic nucleotides, or Ca2+ and/or calmodulin had no significant effect on the reaction, but 20 mg/ml of hemoglobin inhibited the reaction by 60%. -SH-blocking agents but not iodoacetate inhibited the reaction.

Chromatography, Gel↗
PubMed1995

Ultrastructural localization of the C-terminus of the 43-kd dystrophin-associated glycoprotein and its relation to dystrophin in normal murine skeletal myofiber.

We used single and double immunogold labeling electron microscopy to investigate ultrastructural localization of the C terminus of the 43-kd dystrophin-associated glycoprotein (43-DAG) and its relationship to dystrophin in normal murine skeletal myofibers. Single immunolabeling localized the antibody against the C terminus of 43-DAG to the inside surface of the muscle plasma membrane and the sarcoplasmic side of plasma membrane invaginations. Double immunolabeling co-localized antibodies against dystrophin and the C terminus of 43-DAG to the same site noted in the single immunolabeling localization of 43-DAG. In particular, dystrophin and the C-terminal 43-DAG antibody signals were often observed as doublets separated by less than 30 nm. We compared these results with those obtained from double immunogold labeling with anti-dystrophin and anti-beta-spectrin, as well as anti-C-terminal 43-DAG and anti-beta-spectrin antibodies. The antibodies against dystrophin and beta-spectrin, or beta-spectrin and 43-DAG, also co-localized to similar sites in skeletal muscle fibers. Signals of doublet formations were noted but their frequency was significantly lower than the doublet frequency of antidystrophin and anti-43-DAG antibodies. The results support the presence of dystrophin and 43-DAG linkage at the inside surface of the murine skeletal muscle plasma membrane.

Amino Acid Sequence↗
PubMed1994

Red cell membrane disorders in the Japanese population: clinical, biochemical, electron microscopic, and genetic studies.

Based on studies on 610 cases of hereditary red cell membrane disorders, the characteristic features of the incidence of these disorders in the Japanese population are described. These patients were screened by a protocol on red cell morphology (scanning electron microscopy), on red cell membrane proteins (sodium dodecylsulfate polyacrylamide gel electrophoresis, and kinetics of membrane proteins), biophysical studies (ektacytometry, mechanical stability and fluorescence recovery after the photobleaching method), membrane transport (sodium influx and efflux, and anion transport), gene analysis (spectrins, band 4.2 and band 3), surface markers (blood type antigens and sialic acid content), and development and expression of membrane proteins (using a two-phase liquid culture system). Among the molecular abnormalities detected, alpha-spectrin mutation appeared rare (only one family with spectrin alpha I/74), as opposed to two beta-spectrin mutations in Japan out of seven worldwide cases. Two unrelated kindreds with a chromosomal abnormality; that is, del (8) (p11.2-p21.1), were found that involved the possible contribution of ankyrin to the pathogenesis of hereditary spherocytosis. Anomalies of a transmembrane domain of band 3 were detected in two independent kindreds with impaired anion transport. Among 16 HE patients, 13 cases were partially band 4.1 deficient. Complete band 4.2 deficiency of the Nippon type (GCT-->ACT at codon 142 in band 4.2 gene) was observed in 17 cases of 13 unrelated kindreds. Other forms of band 4.2 deficiency without the mutation were also detected in three kindreds. Band 7 deficiency was found in seven cases with hereditary stomatocytosis independent of the presence or absence of cation transport abnormalities. A relatively high incidence of hereditary high red cell membrane phosphatidylcholine hemolytic anemia was disclosed by the analysis of red cell membrane lipids.

Anemia, Hemolytic, Congenital↗
PubMed2000

Stabilization and remodeling of the membrane skeleton during lens fiber cell differentiation and maturation.

Actin filaments are integral components of the plasma membrane-associated cytoskeleton (membrane skeleton) and are believed to play important roles in the determination of cell polarity, shape, and membrane mechanical properties, however the roles of actin regulatory proteins in controlling the assembly, stability, and organization of actin filaments in the membrane skeleton are not well understood. Tropomodulin is a tropomyosin and actin-binding protein that stabilizes tropomyosin-actin filaments by capping their pointed ends and is associated with the spectrin-actin membrane skeleton in erythrocytes, skeletal muscle cells, and lens fiber cells, a specialized epithelial cell type. In this study, we have investigated the role of tropomodulin and other membrane skeleton components in lens fiber cell differentiation and maturation. Our results demonstrate that tropomodulin is expressed concomitantly with lens fiber cell differentiation and assembles onto the plasma membrane only after fiber cells have begun to elongate and form apical-apical contacts with the undifferentiated epithelium. In contrast, other membrane skeleton components, spectrin, actin, and tropomyosin, are constitutively expressed and assembled on the plasma membranes of both undifferentiated and differentiated fiber cells. Tropomodulin, but not other membrane skeleton components, is also enriched at a novel structure at the apical and basal ends of newly elongated fiber cells at the fiber cell-epithelium and fiber cell-capsule interface, respectively. Once assembled, tropomodulin and its binding partners, tropomyosin and actin, remain membrane-associated and are not proteolyzed during fiber cell maturation and aging, despite proteolysis of alpha-spectrin and other cytoskeletal filament systems such as microtubules and intermediate filaments. We propose that actin filament stabilization by tropomodulin, coupled with partial proteolysis of other cytoskeletal components, represents a programmed remodeling of the lens membrane skeleton that may be essential to maintain plasma membrane integrity and transparency of the extremely elongated, long-lived cells of the lens. The unique localization of tropomodulin at fiber cell tips further suggests a new role for tropomodulin at cell-cell and cell-substratum contacts; this may be important for cell migration and/or adhesion during differentiation and morphogenesis.

Actins↗
PubMed1998

Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures.

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 microM), calpain and caspase 3-like proteases processed alpha-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of alpha-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of alpha-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation.

Animals↗
PubMed1994

Erythrocyte membrane vesicles and irreversibly sickled cells bind protein S.

Plasma levels of free protein S, a vitamin K-dependent anticoagulant, are decreased in persons with sickle cell anemia, but the etiology of the low levels is unknown. Protein S binds to phosphatidylserine-containing phospholipids in a calcium-dependent manner. Other studies have indicated that phosphatidylserine may be abnormally present on the outer surface of the membrane lipid bilayer of sickle cells and of the spectrin-depleted vesicles they shed in vivo. We studied the binding of purified, radiolabeled protein S to spectrin-depleted erythrocyte membrane vesicles and to density-separated fractions of sickle and normal erythrocytes. Calcium-dependent binding of protein S occurred with vesicles and with well-aerated dense irreversibly sickled cells, but not with well-aerated sickle discocytes or with normal erythrocytes. These data provide further evidence that phosphatidylserine is abnormally present on the outer surface of spectrin-depleted vesicles and of irreversibly sickled cells. In addition, protein S binding to such sickle membranes in vivo may be responsible, in part, for the decreased levels of free protein S in sickle cell plasma.

Anemia, Sickle Cell↗
PubMed1994

Changes in cytoskeletal proteins and their mRNAs during maturation of human erythroid progenitor cells.

We have used highly purified human early erythroid progenitors to study changes in cytoskeletal proteins during their maturation and terminal differentiation. When erythroid progenitors at the burst-forming unit-erythroid (BFU-E) stage of development are grown in the presence of erythropoietin, the cells mature and terminally differentiate into reticulocytes during a 14-15-day culture period. We have shown by immunofluorescence that spectrin is present in day 3 BFU-E, at which time proteins band 3, ankyrin, and band 4.1 cannot be detected. Ankyrin and band 4.1 were detected in the majority of the cells by day 7 of culture, at the colony-forming unit (CFU)-E stage, whereas only 15% of the cells were positive for band 3 protein on day 7 of culture. The mRNA level for spectrin was already at its maximum on day 8 whereas the mRNAs for band 3, ankyrin, and band 4.1 were just beginning to accumulate. After enucleation, spectrin, band 3, ankyrin, and band 4.1 fluorescence were all associated with the reticulocytes. Actin was localized at the constriction between the extruding nucleus and the incipient reticulocyte in enucleating erythroblasts suggesting a key role for actin in the enucleation of human erythroblasts. Our investigations have also shown that purified human erythroid progenitors cultured in serum-free suspension media are capable of enucleating without the requirement of an extracellular matrix. These results demonstrate that the synthesis and expression of major cytoskeletal proteins in the human erythrocyte membrane occur in an asynchronous manner and that the remodeling of the membrane skeleton begins at a very early stage during erythrocyte development.

Actins↗
PubMed1978

Vesicles isolated from ATP-depleted erythrocytes and out of thrombocyte-rich plasma.

When human erythrocytes are depleted of endogenous ATP they release spectrin-free vesicles as a light vesicle fraction [Lutz et al: J Cell Biol 73: 548, 1977] and chains of rounded vesicles as well as flattened myelin forms in a heavy vesicle fraction. The heavy fraction retains some spectrin, and glycophorin is partially degraded. The release of both types of fragments is not dependent upon added Ca+2, and 50 micrometer EGTA does not prevent the vesicle release. Concomitant with vesicle release, a large fraction of the major protein components of the cell is found in disulfide-bonded aggregates. A protocol is outlined to recover erythrocyte-specific fragments from thrombocyte-rich plasma. It allows detection of spectrin-free vesicles in whole blood stored under blood bank conditions for 12 days. In freshly drawn blood no such vesicles are observed, but particles are obtained that are different from thrombocyte fragments and that show a prominent glycoprotein running slightly faster than glycophorin.

Adenosine Triphosphate↗
PubMed1994

Fetus-like dystrophin expression and other cytoskeletal protein abnormalities in centronuclear myopathies.

We have investigated supposed maturational arrest of muscle in centronuclear myopathies (CNMs) by characterizing the expression of dystrophin, other cytoskeletal proteins, and fetal myosin in the muscle fibers of 9 CNM patients (4 sporadic, 3 familial, 2 adult sporadic). Dystrophin and beta-spectrin localized intracytoplasmically in centrally nucleated fibers. Talin and vinculin were normally expressed. Desmin was radially organized in several fibers in all patients. Scattered vimentinpositive fibers were found in 3 cases. Six myotonic dystrophy cases and 4 inflammatory myopathy cases with regenerating fibers were also studied: dystrophin and the membrane cytoskeletal proteins were normally expressed in the former; and dystrophin, spectrin, and vinculin were reduced in the latter. Intracytoplasmic dystrophin is further evidence of maturational arrest in CNMs. Spectrin and dystrophin codistribute in these pathological conditions as in normal muscle. We conclude that the altered cytoskeletal network found in CNMs likely plays a pathogenetic role in these conditions.

Adult↗
PubMed2002

Short window of opportunity for calpain induced growth cone formation after axotomy of Aplysia neurons.

Our laboratory has established that local activation of calpain by a transient elevation of the free intracellular calcium concentration is crucial for the induction of growth cone (GC) formation in cultured Aplysia neurons. The mechanisms and stages in which calpain is involved in the formation of a GC are not known. We began to study these questions by determining the nature of calpain's action and the stages in which calpain activity affects the cascade of events that leads to the formation of the GC and its extension. We report that the calpain-dependent transformation of an axonal segment into a GC occurs within a narrow window of opportunity that lasts approximately 5 min. If calpain is inhibited during this window of opportunity, GC formation does not occur. Inhibition of calpain after the window of opportunity slows down the rate of lamellipodial extension but doesn't arrest it. The proteolysis of spectrin, a calpain substrate and a major component of the membrane skeleton, occurs within this window of opportunity, in agreement with the hypothesis that spectrin proteolysis is an early step in the formation of the GC. If the onset of proteolysis is deferred, spectrin remains unchanged and GC formation is compromised. We suggest that calpain participates in two different processes: it is critical for the triggering of GC formation and plays a modulatory role during the extension of the GC's lamellipodia.

Animals↗
PubMed1993

Biogenesis of erythrocyte membrane skeleton in health and disease.

To study the biogenesis of red cell membrane skeleton at various stages of erythroid differentiation, we have chosen the following model systems: a) Rauscher erythroleukemia cell line representing the early stages of differentiation, b) Friend erythroleukemia cells, and c) in vitro cultured human erythroblasts. The latter two systems represent terminally differentiated erythroblasts. Using these model systems, we have shown asynchronous synthesis of membrane proteins during erythroid differentiation. At the early stages of erythroid development, the synthesis of spectrin, ankyrin and band 4.1 proteins is initiated before that of the band 3 protein. Following erythroid induction with erythropoietin and dimethylsulfoxide (DMSO), there is a dramatic increase in the synthesis of the band 3 protein without noticeable changes in the synthesis of other membrane proteins. This increase in band 3 synthesis is accompanied by increased stability and recruitment of the skeletal proteins into the membrane skeleton, leading to increased steady state levels. The progressive increase in band 3 synthesis continues during terminal maturation of erythroblasts. This is accompanied by increased stability and assembly of spectrin and ankyrin on the membrane, despite their reduced synthesis. These results point to a key role for the band 3 protein in anchoring and stabilizing these proteins into the permanent skeletal network. Finally, to detect defects of skeletal biosynthesis, we have extended these studies to a patient with severe hereditary spherocytosis characterized by a combined deficiency of spectrin and ankyrin. We have shown that this combined deficiency is a consequence of reduced ankyrin synthesis and mRNA content representing a thalassemia-like membrane protein mutation.

Animals↗