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PubMed1993

A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated.

A tightly membrane-associated form of spectrin (TMA-spectrin) was labeled when human red blood cells were incubated with [3H]palmitic acid. About 90% of spectrin was not fatty acid-acylated and was extracted from membranes by low salt buffers. The 3H-palmitoylated TMA-spectrin, however, resisted low and even high salt extraction and remained associated with inside-out vesicles that were generated in the process of spectrin-actin extraction from membranes. TMA-spectrin was preferentially extracted from KCl-stripped vesicles by 5 M urea at low ionic strength. TMA-spectrin was purified by gel filtration and by ion exchange chromatography in the presence of urea and a non-ionic detergent. Purified TMA-spectrin was 3H-palmitoylated exclusively in the beta subunit to 0.28 mol/mol after a 12-h incubation of red cells. The labeled palmitate may be bound as an ester or thioester, since hydroxylamine (1 M, pH 7.5) released the label completely. Peptide maps of 3H-palmitoylated TMA-spectrin showed three or two labeled peptides from the beta subunit, when generated by V8 protease and trypsin, respectively. Two types of antibodies to spectrin reacted with purified TMA-spectrin, and TMA-spectrin contained the same antigenic peptides as low salt-extractable spectrin. Rabbit anti-ankyrin antibodies did not bind to TMA-spectrin. The substoichiometric incorporation of [3H]palmitic acid into TMA-spectrin could result from the slow turnover of endogenously bound fatty acids. Generation of the tightly membrane-associated and 3H-palmitoylated subpopulation of spectrin cannot be due to entrapment of an unmodified residual fraction of spectrin in right-side-out vesicles. Instead, the data suggest the existence of a subpopulation of spectrin molecules that undergo a covalent fatty acid modification and thereby alter their binding properties. This may offer a new, metabolically dependent mechanism for dynamic interactions between spectrin and the membrane lipid bilayer.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

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 alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-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 [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-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 beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-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 alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-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 alpha- and beta-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.

Animals↗
PubMed1994

A partial structural repeat forms the heterodimer self-association site of all beta-spectrins.

The self-polymerization of alpha beta-spectrin heterodimers to form tetramers and higher oligomers is central to its role as a membrane stabilizer and organizer. Mutations near the amino terminus of alpha I sigma 1-spectrin or the COOH terminus of beta I sigma 1-spectrin often lead to profound impairment heterodimer polymerization and to hemolytic disease of varying severity. Previous studies using an 80-kDa univalent fragment of alpha I sigma 1-spectrin have established that the amino-terminal segment of alpha I sigma 1-spectrin mediates the association of the alpha subunit with either intact heterodimers or with isolated beta-spectrin (beta I sigma 1). However, the nature of the self-association site in beta-spectrin has remained unclear. In the present study, native beta-spectrin and recombinant beta-spectrin peptides representing COOH-terminal portions of two alternative transcripts of the gene on chromosome 2 (beta I sigma 1 or "erythrocyte" spectrin and beta I sigma 2 or "muscle" spectrin), and one transcript of the gene on chromosome 14 (beta II sigma 1 or "beta G-fodrin") have been examined for their ability to bind either intact alpha beta-spectrin or the alpha I-spectrin 80-kDa univalent fragment. Deletion of the nonhomologous beta-spectrin sequence downstream of repeat 17 (spectrin domain III) had no discernible effect on binding. Truncations proximal to codon 2085 of beta I sigma 1-spectrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity. Further truncations to repeat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule. Repeat 16/17 and 17/16 chimeras displayed reduced binding activity. Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation, involves widespread interactions within the 17th repeat unit, is largely independent of sequences in domain III, and can be recreated by the deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units. All beta-spectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.

Amino Acid Sequence↗
PubMed1992

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1984

Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures.

The intracellular precipitation of nonerythrocyte spectrin has been achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the alpha chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells, and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and two proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the two types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. We conclude that the integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape, or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin, however, did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

The lethal hemolytic mutation in beta I sigma 2 spectrin Providence yields a null phenotype in neonatal skeletal muscle.

Point mutations in beta I sigma 1 spectrin that impair the self-association of spectrin alpha beta heterodimers cause mild to severe hemolytic disease and erythrocyte shape abnormalities. Most such mutations act in a dominant negative fashion. One mutation that is particularly devastating is found in beta spectrin Providence. The Providence mutation replaces serine2019 with proline. Heterozygotes display microcytic and fragile erythrocytes; homozygotes die in the neonatal period. It has recently been determined that an alternative transcript of the same beta I sigma 1 spectrin gene expressed in erythroid lineage cells is the major spectrin in skeletal and cardiac muscle and in some neurons. Because the site of the Providence mutation is common to both beta I sigma 1 and beta I sigma 2 spectrin, defective protein must also be expressed in these tissues. Yet the impact of this or any other beta I spectrin mutation outside of the red cell is unexplored. To address this question with respect to skeletal muscle, we have examined the effects of the Providence mutation in cultured muscle cells, after adoptive gene transfer to adult mice, and in two infants homozygous for spectrin Providence. Transfection of the FLAG epitope tagged wild-type beta I sigma 2 or Providence beta I sigma 2 cDNA constructs into C2C12 myoblasts demonstrated by sedimentation velocity analysis that spectrin beta I sigma 2 Providence formed alpha II/-beta I sigma 2 heterodimers in muscle cells but not heterotetramers. Correspondingly, wild-type beta I sigma 2 spectrin formed both alpha II/beta I sigma 1 dimers and heterotetramers, although the proportion of dimers was surprisingly high, which suggested some limitation on self-association in the muscle environment. After adoptive gene transfer into adult mouse skeletal muscle in vivo, both the wild-type and mutant beta I sigma 2 spectrins assembled into a subsarcolemmal complex in a pattern indistinguishable from the native spectrin skeleton. Skeletal muscle taken at autopsy from two infants homozygous for spectrin Providence was normal histologically, as was the intracellular distribution of beta I sigma 2 spectrin as measured by immunoperoxidase staining. These patients also revealed no clinical evidence of myopathy or muscle wasting. It is unknown if they would have experienced dystrophic or myopathic changes if they had lived longer, although we believe that this is unlikely based on the absence of clinical myopathies in patients with other (albeit less severe) beta I spectrin self-association defects. Collectively, these observations indicate that the spectrin mutations that impact tetramer and oligomer formation, even those with a severe hemolytic phenotype, do not impact skeletal muscle function primarily because skeletal muscle does not use the oligomerizing feature of the spectrin skeleton to the same degree as erythrocytes.

Amino Acid Sequence↗
PubMed1985

Expression and assembly of the erythroid membrane-skeletal proteins ankyrin (goblin) and spectrin in the morphogenesis of chicken neurons.

The membrane-skeleton of adult chicken neurons in the cerebellum and optic system is composed of polypeptides structurally and functionally related to the erythroid proteins spectrin and ankyrin, respectively. Neuronal spectrin comprises two distinct complexes that share a common alpha subunit (Mr 240,000) but which have structurally distinct polymorphic subunits (beta' beta spectrin; Mr 220/225,000; gamma spectrin, Mr 235,000); the brain-specific form (alpha gamma spectrin or fodrin) and an erythrocyte-specific form (alpha beta' beta spectrin). Two structurally related isoforms of ankyrin have also been identified and are termed alpha (Mr 260,000) and beta (Mr 237,000) ankyrin. Immunofluorescence demonstrates that the variants of spectrin and ankyrin, respectively, have different distributions within neurons. On the one hand, alpha gamma spectrin and beta ankyrin are present throughout the neuron, in the perikaryon, dendrites, and axon, whereas alpha beta' spectrin and alpha ankyrin are localized exclusively in the perikaryon and dendrites where they are actively segregated from alpha gamma spectrin and other components of axonal transport. This asymmetric distribution of spectrin and ankyrin isoforms is established in distinct stages during neuronal morphogenesis. Early in cerebellar and retinal development, alpha gamma spectrin is expressed in mitotic cells. Subsequently beta ankyrin and alpha gamma spectrin are coexpressed in postmitotic cells and gradually accumulate on the plasma membrane in a uniform pattern throughout the neuron during the phase of cell growth. At the onset of synaptogenesis and the cessation of cell growth, their levels of synthesis decline sharply while the assembled proteins remained as stable membrane components. Concomitantly, there is a dramatic induction in the accumulation of alpha ankyrin and alpha beta' spectrin, whose assembly is limited to the plasma membrane of the perikarya and dendrites. These results demonstrate that two successive, developmentally regulated programs of ankyrin and spectrin expression and patterning on the plasma membrane are involved in the assembly of the spectrin-based asymmetry in the neuronal membrane-skeleton, and that their asymmetric distribution is actively maintained throughout the life of the neuron.

Animals↗
PubMed1985

Some viscoelastic properties of human erythrocyte spectrin networks end-linked in vitro.

We have succeeded in making macroscopic networks of end-linked human erythrocyte spectrin. The network junctions were made using erythrocyte protein 4.1 irreversibly attached to 5 nm (diameter) colloidal gold particles. Rotary shadowing electron microscopy verifies that the protein 4.1-labelled colloidal gold particles bind only to the tail end of the spectrin molecules. Electron micrographs of protein 4.1-labelled colloidal gold particles incubated at 4 degrees C with spectrin dimers reveal that 1-5 spectrin dimers attach to each protein 4.1-labelled colloidal gold particle yielding a spider-like appearance of these complexes. Incubation with a low concentration of spectrin tetramers instead of dimers leads to extensive formation of spectrin microaggregates whereas use of spectrin concentrations higher than 3 mg/ml and a molar ratio between spectrin tetramers and protein 4.1/Au of 4 leads to formation of macroscopic spectrin networks. We have quantitated the viscoelastic properties of such end-linked macroscopic spectrin networks using a gravitational pendulum viscoelastometer. We find that in vitro end-linked spectrin networks can be described by linear viscoelastic theory. The dynamic storage modulus increases almost linearly with the spectrin-protein 4.1/gold particle concentration when the spectrin concentration exceeds about 3 mg/ml and the molar ratio between spectrin tetramers and protein 4.1/Au is 4. At a spectrin concentration of 6 mg/ml and the same ratio between spectrin and protein 4.1/Au, we find a dynamic storage modulus at low frequency of about 80 dyn/cm2. This is in adequate agreement with what is predicted by simple elastomer theory.

Blood Proteins↗
PubMed1986

Biosynthesis of spectrin and its assembly into the cytoskeletal system of Friend erythroleukemia cells.

Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.

Animals↗
PubMed1981

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

Actins↗
PubMed1992

Characterization of human brain cDNA encoding the general isoform of beta-spectrin.

The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and was highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells.

Amino Acid Sequence↗
PubMed1987

Ontogeny, compartmentation, and turnover of spectrin isoforms in rat central neurons.

A variant of a principal structural protein of erythrocytes, spectrin, is a major neuronal protein. Here we have examined the subcellular and regional distributions, subunit composition, ontogeny, and metabolism of spectrin in rat CNS. While all subcellular fractions, except the mitochondrial, expressed the previously characterized brain form of spectrin (fodrin, or alpha gamma-spectrin), limited brain regions contained, in cytoplasm, a second isoform immunologically related to erythrocyte alpha beta-spectrin. Both alpha gamma- and alpha beta-spectrin are primarily neuronal, as evidenced by immunocytochemistry. The spectrins are distributed between 2 distinct subneuronal compartments: a membrane-associated domain containing alpha gamma-spectrin in relatively constant amounts across brain regions, and a cytoplasmic domain containing both the alpha gamma and alpha beta isoforms in widely varying amounts across brain regions. Although forebrain has considerable alpha beta-spectrin, the diencephalon, mesencephalon, and brain stem are devoid of this isoform. Further evidence for spectrin compartmentation comes from its ontogeny. Membrane-associated alpha gamma-spectrin is present at birth at its adult levels, but cytoplasmic alpha beta-spectrin is expressed only following the second postnatal week. Similarly, the 4-fold difference in cytoplasmic alpha gamma-spectrin content across brain regions develops during the third postnatal week. In this compartment, both spectrin forms may be metabolized in vivo, at least in part, by calcium-activated proteolysis. The presence in mammalian neurons of 2 spectrin isoforms and their compartmentation into distinct domains suggests multiple functions for neuronal spectrin, one of which may be in the stabilization or maturation of forebrain neurons.

Aging↗
PubMed1986

Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes.

Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self-associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.

Binding Sites↗
PubMed1990

Patterns of spectrin transcripts in erythroid and non-erythroid cells.

Spectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non-identical spectrins (fodrins) were also found in various non-erythroid tissues. Spectrin mRNA in erythroid and various non-erythroid cells was examined by direct hybridization with human alpha-spectrin, beta-spectrin (erythroid spectrins), and alpha-fodrin (non-erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)+ RNA revealed a distinct pattern of expression in erythroid vs. non-erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of beta-spectrin, whereas they expressed only a single species of alpha-spectrin, and no alpha-fodrin mRNA. In contrast, non-erythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no alpha-spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of alpha-fodrin mRNA. Additionally, they also expressed from none to multiple species of beta-spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non-erythroid spectrin, alpha-fodrin, were found as a single copy only in non-erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to alpha-fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non-erythroid species. These data indicated that several mRNA species of beta-spectrin could be detected in both erythroid and some non-erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogeneity is unclear. However, in each case, the pattern combination observed appeared to be tissue-specific.

Blotting, Northern↗
PubMed1984

Normal content of brain spectrin-like protein in sph/sph mice.

In the erythrocytes of WBB6F1-sph/sph mice spectrin constitutes only approximately 1% of the total sph/sph membrane protein compared to approximately 23% in WBB6F1-+/+ controls. No increase in proteolytic degradation of spectrin in sph/sph erythrocyte membranes could be detected with antibodies directed against mouse erythrocyte spectrin or mouse brain spectrin-like protein. As attachment of normal spectrin to the erythrocyte membrane of these animals appeared to be normal, and as spectrin is not detected when whole sph/sph erythrocytes are solubilized in SDS for SDS PAGE, the deficient erythrocyte spectrin was probably due to diminished production. Brain spectrin-like protein, a nonerythroid spectrin analogue, is antigenically, morphologically and functionally related to erythrocyte spectrin, but appears by peptide mapping analysis to be a distinct gene product. It was found by protein- and antibody-staining of brain membranes to be present in normal concentrations in sph/sph animals. Indirect immunofluorescence of mouse brain tissue with anti-brain spectrin-like protein IgG or anti-erythrocyte spectrin IgG indicated that the distribution of brain spectrin-like protein was normal in sph/sph brain. Therefore the mutation causing diminished production of sph/sph erythrocyte spectrin does not affect the expression of this nonerythroid spectrin analogue.

Animals↗
PubMed1993

The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specific domains of cerebellar neurons.

Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no biochemical restrictions on the assembly of alpha beta heterodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of alpha- and beta-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (beta I sigma 2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell beta-spectrin gene (spectrin beta I sigma 2). In this version, the 3' sequence of erythroid beta-spectrin (beta I sigma 1) is replaced with an alternative sequence that shares substantial homology with the 3' sequence of non-erythroid beta-spectrin (beta II sigma 1). The antibody to beta I sigma 2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with beta II sigma 1 spectrin (beta-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The beta I sigma 2 isoform co-immunoprecipitates with non-erythroid alpha-spectrin (alpha II sigma), even though the distribution of alpha II sigma within neurons only partially overlaps that of beta I sigma 2. No hybrid beta I sigma 2 and beta II sigma 1 (beta-fodrin) spectrin complexes appear to exist. Spectrin beta I sigma 2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of beta I sigma 2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte alpha beta-spectrin antibody. These findings establish the highly precise segregation of a beta-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid alpha-spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1978

Phosphorylation and dephosphorylation of spectrin.

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.

Adenosine Triphosphate↗
PubMed1983

Spectrin phosphorylation in senescent rat erythrocytes.

The rates of phosphorylation and dephosphorylation of the erythrocyte cytoskeletal protein, spectrin, were analyzed in young and old rat erythrocytes. Endogenous membrane protein kinase activity was measured in age-separated rat erythrocytes, and was found to decrease as a function of cell age. Membranes prepared from young and old erythrocytes contained comparable levels of protein phosphatase activity. Spectrin phosphatase activity was readily observed in erythrocyte membranes. Partially purified spectrin kinase and spectrin were prepared from membranes obtained from young and old erythrocytes, and the phosphorylation of the spectrin fractions was measured with the isolated kinases. The kinases prepared from young or old cells phosphorylated spectrin from young cells to the same extent. When spectrin from old cells was used as the substrate, it was phosphorylated ten-fold less extensively by the spectrin kinase prepared from old cells than by the spectrin kinase from young cells. This finding indicated that the decreased phosphorylation of spectrin observed in membranes prepared from age-separated red cells was due to a structural alteration in the spectrin. A structural basis for the decreased phosphorylation of spectrin in older erythrocytes was sought. Treatment of erythrocyte membranes with malonyldialdehyde, a product of lipid peroxidation which accumulates in erythrocyte membranes during senescence, adversely affected spectrin phosphorylation. The results presented here indicate that intramolecular derivatization of spectrin was sufficient to impair its function as a substrate for protein kinase.

Animals↗