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PubMed1984

Oligomeric states of spectrin in normal erythrocyte membranes: biochemical and electron microscopic studies.

We estimated the relative amounts of oligomeric species of spectrin in 0 degrees C red-cell-membrane extracts, including those released from spectrin-actin-polypeptide 4.1 complexes after mild urea treatment. Spectrin dimers, tetramers, and medium-size oligomers were the prominent species, accounting for 5%-10%, 45%-55%, and 25%-35% of spectrin, respectively. When examined by low-angle rotary-shadowing electron microscopy, these medium-size spectrin oligomers (e.g., hexamers, octamers, decamers , dodecamers , and quadecamers ) appeared as polyskelions formed by head-to-head association of three to seven dimers. They were stable species capable of binding to, and subsequent release from, inside-out vesicles without degradation to tetramers or dimers. The data suggest that spectrin tetramers and medium-size oligomers coexist in the normal erythrocyte membrane as the primary native spectrin species.

Chromatography, Gel↗
PubMed1994

Developmental change of alpha-spectrin mRNA in the rat brain.

Spectrin is a cytoskeletal protein considered to be a major component of intracellular cohesion. Using an in situ hybridization approach, we have investigated the developmental expression of the mRNA encoding the alpha-subunit of rat brain spectrins, from birth to adulthood. alpha-Subunit mRNA is detectable at birth, in brain areas with perinatal neurogenesis, such as the cerebral cortex, hippocampus, thalamus, and olfactory bulb. alpha-Brain-spectrin mRNA increases gradually during the first postnatal days to reach a plateau between the second and the third week of life. In the young adult brain, the level of alpha-brain spectrin mRNA decreased globally. This spacio-temporal distribution argues for the involvement of the mRNA in the synthesis of both the erythroid and non-erythroid brain spectrin isoforms. We have focused our attention on the hippocampal formation and the cerebellum. In both regions, in situ hybridization signal variations are superimposable with neuronal maturation gradients. This pattern of variation, coupled with the known interaction of brain spectrins with other cytoskeletal proteins, agrees with the notion that brain spectrins may be involved in neuronal differentiation by way of the cytoskeletal lattice organization.

Animals↗
PubMed1985

Mechanism of cytoskeletal regulation (I): functional differences correlate with antigenic dissimilarity in human brain and erythrocyte spectrin.

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the alpha-subunit (alpha-I and alpha-III) and of terminal portions of the 28 kDa and 74 kDa domains of the beta-subunit (beta-I and beta-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.

Brain Chemistry↗
PubMed1985

Structural and functional analysis of spectrin from neonatal erythrocytes.

Spectrin was purified by rate zonal sedimentation from low-salt extracts of red cell membranes from neonatal and adult blood. Neonatal and adult spectrin cosedimented in sucrose density gradients, comigrated on SDS gels and displayed identical two-dimensional chymotryptic 125I-labelled peptide maps. Neonatal spectrin and adult spectrin exhibited equivalent affinity for both neonatal and adult ankyrin sites on spectrin-depleted inverted membrane vesicles. Purified spectrin heterodimers from neonatal and adult red cells displayed similar self-association equilibrium constants in a fluid phase dimer-dimer association assay. These results suggest that the unique membrane characteristics of the neonatal erythrocyte are not due to a structural or functional alteration of spectrin. Several alternative hypotheses involving other membrane proteins and their linkages are discussed.

Centrifugation, Density Gradient↗
PubMed1986

Spectrin expression in neuroblastoma cells.

Mouse neuroblastoma cells express a spectrin-related molecule containing 240 kDal (kiloDalton) and 235 kDal subunits in a 1:1 ratio. The 240 kDal and 235 kDal subunits are nearly identical to the alpha and beta subunits respectively of brain spectrin by two dimensional chymotryptic peptide mapping analysis. The neuroblastoma cells do not express a measureable quantity of a red blood cell (rbc)-type spectrin molecule. Neuroblastoma spectrin has been localized throughout the cell body, and neurites of these cells by indirect immunofluorescence studies. As neuroblastoma cells are homogeneous, neuron-like, available in large quantity, and synthesize a single variant of spectrin which is closely related to brain spectrin(240/235), it is the best available model system for the study of the synthesis, assembly and turnover of a neuronal spectrin subtype.

Animals↗
PubMed1995

Brain spectrin: of mice and men.

This article reviews our current knowledge of the structure of alpha spectrins and beta spectrins in the brain, as well as their location and expression within neural tissue. We discuss the known protein interactions of brain spectrin isoforms, and then describe results that suggest an important role for spectrin (alpha SpII sigma 1/beta SpII sigma 1) in the Ca(2+)-regulated release of neurotransmitters. Evidence that supports a role for spectrin in the docking of synaptic vesicles to the presynaptic plasma membrane and as a Ca2+ sensor protein that unclamps the fusion machinery is described, along with the Casting the Line model, which summarizes the information. We finish with a discussion of the value of spectrin and ankyrin-deficient mouse models in deciphering spectrin function in neural tissue.

Amino Acid Sequence↗
PubMedDate not supplied

Spectrin properties and the elasticity of the red blood cell membrane skeleton.

Two models of spectrin elasticity are developed and compared to experimental measurements of the red blood cell (RBC) membrane shear modulus through the use of an elastic finite element model of the RBC membrane skeleton. The two molecular models of spectrin are: (i) An entropic spring model of spectrin as a flexible chain. This is a model proposed by several previous authors. (ii) An elastic model of a helical coiled-coil which expands by increasing helical pitch. In previous papers, we have computed the relationship between the stiffness of a single spectrin molecule (K) and the shear modulus of a network (mu), and have shown that this behavior is strongly dependent upon network topology. For realistic network models of the RBC membrane skeleton, we equate mu to micropipette measurements of RBCs and predict K for spectrin that is consistent with the coiled-coli molecular model. The value of spectrin stiffness derived from the entropic molecular model would need to be at least 30 times greater to match the experimental results. Thus, the conclusion of this study is that a helical coiled-coil model for spectrin is more realistic than a purely entropic model.

Cytoskeleton↗
PubMed2000

States and transitions during forced unfolding of a single spectrin repeat.

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so-called spectrin repeats. Each repeat contains three antiparallel alpha-helices that form a coiled-coil structure. Spectrin forms an oligomeric structure that is able to cross-link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force-extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.

Animals↗
PubMed2002

The spectrin repeat: a structural platform for cytoskeletal protein assemblies.

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.

Actinin↗
PubMed2005

Spectrin, alpha-actinin, and dystrophin.

Spectrin family proteins represent an important group of actin-bundling and membrane-anchoring proteins found in diverse structures from yeast to man. Arising from a common ancestral alpha-actinin gene through duplications and rearrangements, the family has increased to include the spectrins and dystrophin/utrophin. The spectrin family is characterized by the presence of spectrin repeats, actin binding domains, and EF hands. With increasing divergence, new domains and functions have been added such that spectrin and dystrophin also contain specialized protein-protein interaction motifs and regions for interaction with membranes and phospholipids. The acquisition of new domains also increased the functional complexity of the family such that the proteins perform a range of tasks way beyond the simple bundling of actin filaments by alpha-actinin in S. pombe. We discuss the evolutionary, structural, functional, and regulatory roles of the spectrin family of proteins and describe some of the disease traits associated with loss of spectrin family protein function.

Actinin↗
PubMed1997

Correlation of ischemia/reperfusion or partial outlet obstruction-induced spectrin proteolysis by calpain with contractile dysfunction in rabbit bladder.

OBJECTIVES: In the rabbit, both experimental ischemia and partial outlet obstruction of the urinary bladder induce similar dysfunctions with regard to the contractile responses to both field (neuronal) stimulation and postsynaptic receptor stimulation. Circumstantial evidence indicates that the pathologic response to both conditions is related to two connected processes-tissue ischemia and reperfusion injury-that result in a marked increase in intracellular calcium ([Ca2+]i), followed by the activation of the Ca(2+)-dependent neutral protease calpain. Calpain activation results in the proteolysis of specific membrane proteins, including those of neuronal membranes (resulting in progressive denervation of the detrusor) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), resulting in the previously reported decrease in SERCA. The current study is designed to generate direct support for the theory that both ischemia and partial outlet obstruction result in the activation of calpain. METHODS: Separate sets of rabbits were subjected to 1 or 2 hours of ischemia, followed by reperfusion for different lengths of time, or partial outlet obstruction for different lengths of time. We determined the state of calpain activation by quantitating tissue proteolysis of alpha-spectrin by Western blot analysis. Correlative organ bath studies were conducted to observe the contractile responses of bladder strips to field stimulation and bethanechol administration. RESULTS: (1) Sixty minutes of ischemia followed by 30 minutes of reperfusion resulted in (a) a reduction in the contractile responses to field stimulation and bethanechol (89% and 57%, respectively), and (b) a 72% decrease in native alpha-spectrin, with a concomitant 300% increase in its breakdown products (BDPs). Neither alpha-spectrin nor its BDPs had returned to control levels after 72 hours of reperfusion. (2) Twenty-four hours after the creation of a partial obstruction, alpha-spectrin BDP levels were increased 330%, then gradually fell to 130% of control levels by 14 days after obstruction. Concomitantly, the native alpha-spectrin level was decreased 74% 24 hours after obstruction and remained low through 7 days after obstruction. At 14 days after obstruction, the alpha-spectrin levels had recovered to 75% of control levels. CONCLUSIONS: These findings suggest that Ca(2+)-dependent proteolysis of the preferred calpain substrate alpha-spectrin in urinary bladder tissues is increased significantly by both ischemia/reperfusion and partial outlet obstruction. Temporally, proteolysis precedes the reduced muscle function resulting from these pathologic conditions.

Animals↗
PubMed2001

Spectrins in developing rat hippocampal cells.

We studied the spectrins in developing hippocampal tissue in vivo and in vitro to learn how they contribute to the organization of synaptic and extrasynaptic regions of the neuronal plasma membrane. beta-Spectrin, but not beta-fodrin or alpha-fodrin, increased substantially during postnatal development in the hippocampus, where it was localized in neurons but not in astrocytes. Immunoprecipitations from neonatal and adult hippocampal extracts suggest that while both beta-spectrin and beta-fodrin form heteromers with alpha-fodrin, oligomers containing all three subunits are also present. At the subcellular level, beta-fodrin and alpha-fodrin were present in the cell bodies, dendrites, and axons of pyramidal-like neurons in culture, as well as in astrocytes. beta-Spectrin, by contrast, was absent from axons but present in cell bodies and dendrites, where it was organized in a loose, membrane-associated meshwork that lacked alpha-fodrin. A similar meshwork was also apparent in pyramidal neurons in vivo. At some dendritic spines, alpha-fodrin was present in the necks but not in the heads, whereas beta-spectrin was present at significant levels in the spine heads. The presence of significant amounts of beta-spectrin without an accompanying alpha-fodrin subunit was confirmed by immunoprecipitations from extracts of adult hippocampus. Our results suggest that the spectrins in hippocampal neurons can assemble to form different membrane-associated structures in distinct membrane domains, including those at synapses.

Age Factors↗
PubMed1976

Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.

Actins↗
PubMedDate not supplied

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis.

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.

Erythrocytes↗
PubMedDate not supplied

Modulation of spectrin-actin assembly by erythrocyte adducin.

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.

Actins↗
PubMed2004

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the alpha beta spectrin self-association binding site.

We studied an infant with severe neonatal hemolytic anemia and hyperbilirubinemia that evolved into a partially compensated ellipto-poikilocytic anemia. His father had typical elliptocytosis. Their erythrocyte membranes demonstrated structural and functional defects in spectrin. Genetic studies revealed that the proband and his father were heterozygous for an alpha-spectrin mutation, Ile24Thr, in the alpha beta spectrin self-association binding site. The proband also carried the low expression allele alpha(LELY) in trans, influencing the clinical phenotype. The importance of isoleucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all alpha spectrins from Drosophila to humans. Molecular modeling demonstrated that replacement of a hydrophobic isoleucine with a hydrophilic threonine disrupts highly conserved hydrophobic interactions in the interior of the spectrin triple helix critical for spectrin function.

Binding Sites↗
PubMed1998

Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin.

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.

Actins↗
PubMed2000

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid alpha-spectrin gene.

INTRODUCTION: Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. MATERIALS AND METHODS: Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. RESULTS: Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. CONCLUSION: These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

Alleles↗