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PubMed1991

The Trypanosoma brucei cytoskeleton: ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy.

We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quick-freezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction wit the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.

Animals↗
PubMed2005

Spectrins and the Golgi.

Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.

Animals↗
PubMed2006

Structural cooperativity in spectrin type repeats motifs of dystrophin.

Dystrophin is a member of the spectrin family of proteins, which are characterized as being predominantly composed the spectrin-type-repeat, a triple alpha-helical bundle motif present in multiple tandem copies, producing a rod-like shape. Whether or not this motif, which is determined by sequence homology, is correlated with biophysical domains in the intact protein is uncertain. The nature of the domain structure impacts the flexibility and shape of the rod region of this protein, which is a target for modification in several therapeutic approaches aimed at Duchenne Muscular Dystrophy, a common and fatal genetic disease caused by defective dystrophin. We examined three such motifs in dystrophin, expressing them recombinantly both singly and in tandem, and studying their thermodynamic properties by solvent and thermal denaturation. We have found that the degree to which they are independently stable and expressible varies considerably. The fourth motif appears to be largely stable and independent, whereas the third and second motifs interact strongly.

Amino Acid Motifs↗
PubMed2006

Spectrin-phospholipid interactions. Existence of multiple kinds of binding sites?

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

Binding Sites↗
PubMed2006

Heavy neurofilament accumulation and alpha-spectrin degradation accompany cerebellar white matter functional deficits following forebrain fluid percussion injury.

Evidence for diffuse traumatic axonal injury (TAI) in clinical cases and animal models of traumatic brain injury (TBI) indicate that pathophysiological mechanisms extend to regions remote from the injury epicenter. The potential for indirect cerebellar trauma contributing to TBI pathophysiology is of significance since impairment of motor function and coordination is a common consequence of TBI but is also a domain associated with cerebellar function. The relationship between cerebellar white matter structure and function following traumatic head injury has not been examined. Using the fluid percussion injury (FPI) device applied unilaterally in the forebrain, evoked compound action potential (CAP) recordings from cerebellar white matter of Sprague-Dawley rats indicated a spatial and temporal pattern of electrophysiological deficits throughout the cerebellar vermis. The posterior and middle lobules of the cerebellum exhibited significant declines in evoked CAP amplitude compared to sham controls (p=0.004, p=0.005, respectively). Duration of the CAP decay also increased, suggesting that functional white matter deficits were a combination of axonal loss and compromised axonal integrity. Functional white matter deficits persisted at 14 days post-injury in the posterior and middle regions of the cerebellum. Evidence of heavy chain neurofilament (NF200) degradation was observed at 1 day post-injury by Western blot. Immunohistochemistry labeling for NF200 indicated the presence of highly immunoreactive NF200 axonal swellings consistent with morphological features of TAI. alpha-Spectrin degradation was also observed between 1 and 14 days post-injury. This study demonstrates the electrophysiological consequences of cerebellar white matter injury and a temporal profile of NF200 and spectrin degradation following forebrain FPI.

Action Potentials↗
PubMed2004

The folding of spectrin domains I: wild-type domains have the same stability but very different kinetic properties.

The study of proteins with the same architecture, but different sequence has proven to be a valuable tool in the protein folding field. As a prelude to studies on the folding mechanism of spectrin domains we present the kinetic characterisation of the wild-type forms of the 15th, 16th, and 17th domains of chicken brain alpha-spectrin (referred to as R15, R16 and R17, respectively). We show that the proteins all behave in a two-state manner, with different kinetic properties. The folding rate varies remarkably between different members, with a 5000-fold variation in folding rate and 3000-fold variation in unfolding rate seen for proteins differing only 1 kcal mol(-1) in stability. We show clear evidence for significant complexity in the energy landscape of R16, which shows a change in amplitude outside the stopped-flow timescale and curvature in the unfolding arm of the chevron plot. The accompanying paper describes the characterisation of the folding pathway of this domain.

Animals↗
PubMed2006

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

Amino Acid Sequence↗
PubMed2005

Activation of calpains, calpastatin and spectrin cleavage in the brain during the pathology of fatal murine cerebral malaria.

Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.

Animals↗
PubMed2006

Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach.

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.

Animals↗
PubMed1984

Identification of spectrin and protein 4.1-like proteins in mammalian lens.

Human, bovine, canine, and rabbit lenses were found to contain proteins which cross-react with anti-4.1 serum and which have molecular weights similar to erythrocyte proteins 4.1a and 4.1b (approximately 80 kd). Additionally, bovine, canine, and rabbit lenses contain a 4.1-like protein of approximately 125 kd which is absent from human lens. Proteins which cross-react with antibody to human erythrocyte spectrin were also detected. The human lens showed weak cross-reaction of bands of 240 kd and 225 kd, and a more intense cross-reaction of a band of 235 kd. Canine and bovine lenses showed weak cross-reaction with only the bands at 240 kd and 235 kd. The lens 240 kd band of all species also demonstrated calcium-dependent binding of calmodulin. Our results indicate that proteins related to, but distinct from, erythrocyte protein 4.1 and spectrin are found in mammalian lens.

Animals↗
PubMed1985

Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase.

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.

Actins↗
PubMed2001

Deformation-enhanced fluctuations in the red cell skeleton with theoretical relations to elasticity, connectivity, and spectrin unfolding.

To assess local elasticity in the red cell's spectrin-actin network, nano-particles were tethered to actin nodes and their constrained thermal motions were tracked. Cells were both immobilized and controllably deformed by aspiration into a micropipette. Since the network is well-appreciated as soft, thermal fluctuations even in an unstressed portion of network were expected to be many tens of nanometers based on simple equipartition ideas. Real-time particle tracking indeed reveals such root-mean-squared motions for 40-nm fluorescent beads either tethered to actin directly within a cell ghost or connected to actin from outside a cell via glycophorin. Moreover, the elastically constrained displacements are significant on the scale of the network's internodal distance of approximately 60-80 nm. Surprisingly, along the aspirated projection-where the network is axially extended by as much as twofold or more-fluctuations in the axial direction are increased by almost twofold relative to motions in the unstressed network. The molecular basis for such strain softening is discussed broadly in terms of force-driven transitions. Specific considerations are given to 1) protein dissociations that reduce network connectivity, and 2) unfolding kinetics of a localized few of the red cell's approximately 10(7) spectrin repeats.

Actins↗
PubMed1992

Analysis of the three-alpha-helix motif in the spectrin superfamily of proteins.

Members of the spectrin superfamily of proteins contain different numbers of homologous repeats arranged in tandem. Each of these consists of a three-alpha-helix motif, comprising two similarly and one oppositely directed alpha-helical segment joined by nonhelical linkers of characteristic length. The right-handed alpha-helices each display a heptad repeat in their amino acid sequences indicative of left-handed coiled-coil-like packing. We have calculated the potential number of inter-helix ionic interactions that specify the spatial arrangement of the helices in the motif in terms of both the handedness of helix connectivity (left or right) and the relative axial stagger between the three alpha-helices. All of the models examined were constrained to have optimal coiled-coil packing. For alpha-spectrin and alpha-actinin the results provide strong support for a left-handed connectivity of the three helices and axial repeat lengths of 5.05 and 6.24 nm, respectively. Furthermore, the axial staggers between homologous segments in the preferred models are identical. The insights provided into the topography of this widespread tertiary fold may prove of value to those concerned with the problem of de novo protein design.

Actinin↗
PubMed1996

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.

Actins↗
PubMed1992

Localization of proteins immunologically related to erythrocyte protein 4.1, spectrin and ankyrin in thyroid gland.

Analogues of erythrocyte protein 4.1, spectrin and ankyrin were examined in the thyroid gland of pig and rat by immunohistochemical techniques. Analysis with immunofluorescence microscopy revealed that the peripheral cytoplasm and apical-lateral plasma membrane of follicle epithelial cells of thyroid glands were stained with antibodies against erythrocyte protein 4.1, spectrin, or ankyrin. The results indicate that membrane skeletal protein lattice might exist in thyroid follicle epithelial cells.

Animals↗
PubMed1998

A Plasmodium chabaudi protein contains a repetitive region with a predicted spectrin-like structure.

cDNA and genomic DNA clones covering the entire open reading frame (ORF) for a Plasmodium chabaudi 96V protein were isolated. From the first ATG codon the intronless gene codes for a 229-kDa protein. Antisera raised against recombinant polypeptides coded by two different regions of the gene reacted with a 240/225-kDa doublet on Western blots of parasite extracts. In immunofluorescence studies the same sera detected the antigen at the apical end of the merozoite, possibly in rhoptry organelles. In Western blotting experiments the recombinant polypeptides were recognised by antibodies induced by natural infection. A 364-amino acid residue repetitive region, based on 32 11-mer repeats divided by two 6-mer repeats into three blocks, is located in the centre of the protein. Analysis of this repetitive region led us to propose a model in which each of the three units forms an alpha-helical coiled-coil triple-helix containing a possible leucine-histidine zipper. Each unit resembles in structure the units present in spectrin. The repeat region is flanked by predicted heptad based alpha-helical coiled-coil regions, and we propose that the protein forms a dimer. The 229-kDa protein has the overall character of a cytoskeletal protein. We have named the 229-kDa protein repetitive organellar protein (ROPE) and suggest that ROPE may be involved in the process of invasion, possibly by interacting with the erythrocyte cytoskeleton, and that the leucine histidine-zipper may be involved in molecular mimicry of spectrin.

Amino Acid Sequence↗
PubMed1993

Association of spectrin with manchette microtubules in mammalian spermatids.

In hamster and mouse spermatozoa a spectrin immunogold labeling was found under the plasma membrane in the principal piece of the flagellum. During spermatid differentiation, the spectrin labeling was associated with the manchette, a transient microtubular network involved in nuclear shaping and organelle translocation.

Animals↗
PubMed1994

Spectrin motif. Conformation of a mammoth protein.

The crystal structure of the three-helix-bundle spectrin motif provides new insights into the structure of the large proteins that constitute the spectrin superfamily, which includes dystrophin and alpha-actinin.

Actinin↗