Avian lens spectrin: subunit composition compared with erythrocyte and brain spectrin

Chicken lens spectrin is composed predominantly of equimolar amounts of two polypeptides with solubility properties similar, but not identical, to erythrocyte spectrin. The larger polypeptide, Mr 240,000 (lens alpha- spectrin), co-migrates with erythrocyte and brain alpha-spectrin on one- and two-dimensional SDS polyacrylamide gels and cross-reacts with antibodies specific for chicken erythrocyte alpha-spectrin; the smaller polypeptide, Mr 235,000 (lens gamma-spectrin), co-migrates with brain gamma-spectrin and does not cross-react with either the alpha-spectrin antibodies specific for chicken erythrocyte beta-spectrin. Minor amounts of polypeptides antigenically related to erythrocyte beta- spectrin with a greater electrophoretic mobility than lens gamma- spectrin are also detected in lens. The equimolar ratio of lens alpha- and gamma-spectrin is invariantly maintained during the extraction of lens plasma membranes under different conditions, or after immunoprecipitation of whole extracts of lens with erythrocyte alpha- spectrin antibodies. Two-dimensional peptide mapping reveals that whereas alpha-spectrins from chicken erythrocytes, brain, and lens are highly homologous, the gamma-spectrins, although related, have some cell-type-specific peptides and are substantially different from erythrocyte beta-spectrin. Thus, the expression of cell-type-specific gamma- and beta-spectrins may be the basis for the assembly of a spectrin-plasma membrane complex whose molecular composition is tailored to the functional requirements of the particular cell-type

Biogenesis of the Avian Erythroid Membrane Skeleton : Receptor-mediated Assembly and Stabilization of Ankyrin (Goblin) and Spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the αß-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the ß-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin (goblin) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton . In contrast, continuous labeling of erythroid cells with [(^35)S]methionine, and immunoprecipitation of ankyrin and α- and ß- spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions . The soluble pools of ankyrin and ß-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of α-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of α- to ß-spectrin. Ankyrin and ß-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although α-spectrin enters the cytoskeletal fraction with similar kinetics to ß-spectrin and ankyrin, and in amounts equimolar to ß-spectrin, the amount of cytoskeletal α-spectrin does not exceed the amount of soluble α-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and α- and ß-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and α- and ß-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization

Posttranslational control of membrane-skeleton (ankyrin and alpha beta- spectrin) assembly in early myogenesis

Adult chicken skeletal muscle cells express polypeptides that are antigenically related to alpha-spectrin (Mr 240,000) and beta-spectrin (Mr 220,000-225,000), the major components of the erythrocyte membrane- skeleton, and to ankyrin (Mr 237,000; also termed goblin in chicken erythrocytes), which binds spectrin to the transmembrane anion transporter in erythrocytes. Comparative immunoblotting of SDS- solubilized extracts of presumptive myoblasts and fully differentiated myotubes cultured in vitro demonstrated that there is a dramatic accumulation of ankyrin and alpha- and beta-spectrin during myogenesis and a concomitant switch in the subunit composition of spectrin from alpha gamma to alpha beta. Analysis of early time points in myogenesis (12-96 h) revealed that these changes occur shortly after the main burst of cell fusion. To determine the temporal relationship between cell fusion and the accumulation of ankyrin and alpha- and beta- spectrin, we treated presumptive myoblasts with 2 mM EGTA, which resulted in the complete inhibition of cell fusion. The incorporation of [35S]methionine into total protein and, specifically, into alpha-, gamma-, and beta-spectrin remained the same in EGTA-treated and control cells. Analysis by immunoblotting of the amounts of ankyrin and alpha- and beta-spectrin in fusion-blocked cells revealed that there was no effect on accumulation for the first 19 h. However, there was then a dramatic cessation in their accumulation, and thereafter, the amount of each protein at steady state remained constant. Upon release from the EGTA block, the cells fused rapidly (less than 11 h), and the accumulation of ankyrin and alpha- and beta-spectrin was reinitiated after a lag period of 3-5 h at a rate similar to that in control cells. The inhibition in the accumulation of newly synthesized ankyrin, alpha- spectrin, and beta-spectrin in EGTA-treated myoblasts was not characteristic of all structural proteins, since the accumulation of the muscle-specific intermediate filament protein desmin was the same in control and fusion-blocked cells. These results show that in myogenesis, the synthesis of ankyrin and alpha- and beta-spectrin and their accumulation as a complex, although concurrent, are not coupled events. We hypothesize that the extent of assembly of these components of the membrane-skeleton in muscle cells is determined by a control mechanism(s) operative at the posttranslational level that is triggered near the time of cell fusion and the onset of terminal differentiation

Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton

The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally

Tissue-specific Expression of Distinct Spectrin and Ankyrin Transcripts in Erythroid and Nonerythroid Cells

cDNA probes for three components of the erythroid membrane skeleton, α spectrin, β spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a λgt11 expression vector library containing cDNA prepared from chicken erythroid poly(A)^+ RNA. Each cDNA appears to hybridize to one gene type in the chicken genome. Qualitatively distinct RNA species in myogenic and erythroid cells are detected for β spectrin and ankyrin, while α spectrin exists as a single species of transcript in all tissues examined. This tissue-specific expression of RNAs is regulated quantitatively during myogenesis in vitro, since all three accumulate only upon myoblast fusion. Furthermore, RNAs for two of the three genes do not accumulate to detectable levels in chicken embryo fibroblasts, demonstrating that their accumulation can be noncoordinate. These observations suggest that independent gene regulation and tissue-specific production of heterogeneous transcripts from the β spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells

A lymphoma plasma membrane-associated protein with ankyrin-like properties.

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities

Glial βii spectrin contributes to paranode formation and maintenance

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes intheCNS. Paranodal junctions contribute to both no deassembly and maintenance. Despitetheir importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. βII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell βII spectrin is highly enriched at paranodes. To elucidate the roles of glial βII spectrin, we generated mutant mice lacking βII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions.Fil: Susuki, Keiichiro. Baylor College of Medicine; Estados UnidosFil: Zollinger, Daniel R.. Baylor College of Medicine; Estados UnidosFil: Chang, Kae Jiun. Baylor College of Medicine; Estados UnidosFil: Zhang, Chuansheng. Baylor College of Medicine; Estados UnidosFil: Huang, Claire Yu Mei. Baylor College of Medicine; Estados UnidosFil: Tsai, Chang Ru. Baylor College of Medicine; Estados UnidosFil: Galiano, Mauricio Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Baylor College of Medicine; Estados UnidosFil: Liu, Yanhong. Baylor College of Medicine; Estados UnidosFil: Benusa, Savannah D.. Virginia Commonwealth University; Estados UnidosFil: Yermakov, Leonid M.. Wright State University; Estados UnidosFil: Griggs, Ryan B.. Wright State University; Estados UnidosFil: Dupree, Jeffrey L.. Virginia Commonwealth University; Estados UnidosFil: Rasband, Matthew N.. Baylor College of Medicine; Estados Unido

Stiffening of Red Blood Cells Induced by Disordered Cytoskeleton Structures: A Joint Theory-experiment Study

The functions and elasticities of the cell are largely related to the structures of the cytoskeletons underlying the lipid bi-layer. Among various cell types, the Red Blood Cell (RBC) possesses a relatively simple cytoskeletal structure. Underneath the membrane, the RBC cytoskeleton takes the form of a two dimensional triangular network, consisting of nodes of actins (and other proteins) and edges of spectrins. Recent experiments focusing on the malaria infected RBCs (iRBCs) showed that there is a correlation between the elongation of spectrins in the cytoskeletal network and the stiffening of the iRBCs. Here we rationalize the correlation between these two observations by combining the worm-like chain (WLC) model for single spectrins and the Effective Medium Theory (EMT) for the network elasticity. We specifically focus on how the disorders in the cytoskeletal network affect its macroscopic elasticity. Analytical and numerical solutions from our model reveal that the stiffness of the membrane increases with increasing end-to-end distances of spectrins, but has a non-monotonic dependence on the variance of the end-to-end distance distributions. These predictions are verified quantitively by our AFM and micropipette aspiration measurements of iRBCs. The model may, from a molecular level, provide guidelines for future identification of new treatment methods for RBC related diseases, such as malaria infection.Comment: 8 pages, 4 figures; 3 supporting figure