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1
Machnicka, Beata, Renata Grochowalska, Dżamila M. Bogusławska, and Aleksander F. Sikorski. "The role of spectrin in cell adhesion and cell–cell contact." Experimental Biology and Medicine 244, no. 15 (June 21, 2019): 1303–12. http://dx.doi.org/10.1177/1535370219859003.
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Spectrins are proteins that are responsible for many aspects of cell function and adaptation to changing environments. Primarily the spectrin-based membrane skeleton maintains cell membrane integrity and its mechanical properties, together with the cytoskeletal network a support cell shape. The occurrence of a variety of spectrin isoforms in diverse cellular environments indicates that it is a multifunctional protein involved in numerous physiological pathways. Participation of spectrin in cell–cell and cell–extracellular matrix adhesion and formation of dynamic plasma membrane protrusions and associated signaling events is a subject of interest for researchers in the fields of cell biology and molecular medicine. In this mini-review, we focus on data concerning the role of spectrins in cell surface activities such as adhesion, cell–cell contact, and invadosome formation. We discuss data on different adhesion proteins that directly or indirectly interact with spectrin repeats. New findings support the involvement of spectrin in cell adhesion and spreading, formation of lamellipodia, and also the participation in morphogenetic processes, such as eye development, oogenesis, and angiogenesis. Here, we review the role of spectrin in cell adhesion and cell–cell contact.Impact statementThis article reviews properties of spectrins as a group of proteins involved in cell surface activities such as, adhesion and cell–cell contact, and their contribution to morphogenesis. We show a new area of research and discuss the involvement of spectrin in regulation of cell–cell contact leading to immunological synapse formation and in shaping synapse architecture during myoblast fusion. Data indicate involvement of spectrins in adhesion and cell–cell or cell–extracellular matrix interactions and therefore in signaling pathways. There is evidence of spectrin’s contribution to the processes of morphogenesis which are connected to its interactions with adhesion molecules, membrane proteins (and perhaps lipids), and actin. Our aim was to highlight the essential role of spectrin in cell–cell contact and cell adhesion.
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Goodman, Steven R., Daniel Johnson, Steven L. Youngentob, and David Kakhniashvili. "The Spectrinome: The Interactome of a Scaffold Protein Creating Nuclear and Cytoplasmic Connectivity and Function." Experimental Biology and Medicine 244, no. 15 (September 4, 2019): 1273–302. http://dx.doi.org/10.1177/1535370219867269.
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We provide a review of Spectrin isoform function in the cytoplasm, the nucleus, the cell surface, and in intracellular signaling. We then discuss the importance of Spectrin’s E2/E3 chimeric ubiquitin conjugating and ligating activity in maintaining cellular homeostasis. Finally we present spectrin isoform subunit specific human diseases. We have created the Spectrinome, from the Human Proteome, Human Reactome and Human Atlas data and demonstrated how it can be a useful tool in visualizing and understanding spectrins myriad of cellular functions. Impact statement Spectrin was for the first 12 years after its discovery thought to be found only in erythrocytes. In 1981, Goodman and colleagues 1 found that spectrin-like molecules were ubiquitously found in non-erythroid cells leading to a great multitude of publications over the next thirty eight years. The discovery of multiple spectrin isoforms found associated with every cellular compartment, and representing 2-3% of cellular protein, has brought us to today’s understanding that spectrin is a scaffolding protein, with its own E2/E3 chimeric ubiquitin conjugating ligating activity that is involved in virtually every cellular function. We cover the history, localized functions of spectrin isoforms, human diseases caused by mutations, and provide the spectrinome: a useful tool for understanding the myriad of functions for one of the most important proteins in all eukaryotic cells.
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Moorthy, Suraj, Lihsia Chen, and Vann Bennett. "Caenorhabditis elegans β-G Spectrin Is Dispensable for Establishment of Epithelial Polarity, but Essential for Muscular and Neuronal Function." Journal of Cell Biology 149, no. 4 (May 15, 2000): 915–30. http://dx.doi.org/10.1083/jcb.149.4.915.
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The Caenorhabditis elegans genome encodes one α spectrin subunit, a β spectrin subunit (β-G), and a β-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans α spectrin is reproduced by tandem depletion of both β-G and β-H spectrins. We propose that α spectrin combines with the β-G and β-H subunits to form α/β-G and α/β-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode β-G spectrin and vertebrate β spectrins exhibit three striking parallels including: (1) β spectrins are associated with the sites of cell–cell contact in epithelial tissues; (2) the highest levels of β-G spectrin occur in the nervous system; and (3) β spec-trin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode β-G spectrin associates with plasma membranes at sites of cell–cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode β-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, β-G spectrin is not associated with internal membranes and depletion of β-G spectrin was not associated with any detectable defects in secretion. Instead β-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans β-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.
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Nicolas, Gaël, Catherine M. Fournier, Colette Galand, Laurence Malbert-Colas, Odile Bournier, Yolande Kroviarski, Monique Bourgeois, et al. "Tyrosine Phosphorylation Regulates Alpha II Spectrin Cleavage by Calpain." Molecular and Cellular Biology 22, no. 10 (May 15, 2002): 3527–36. http://dx.doi.org/10.1128/mcb.22.10.3527-3536.2002.
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ABSTRACT Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of αII-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The αII-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the αI-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in αII-spectrin. Western blotting showed that αII-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of αII-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.
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Leto, T. L., D. Fortugno-Erikson, D. Barton, T. L. Yang-Feng, U. Francke, A. S. Harris, J. S. Morrow, V. T. Marchesi, and E. J. Benz. "Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species." Molecular and Cellular Biology 8, no. 1 (January 1988): 1–9. http://dx.doi.org/10.1128/mcb.8.1.1-9.1988.
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The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
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Leto, T. L., D. Fortugno-Erikson, D. Barton, T. L. Yang-Feng, U. Francke, A. S. Harris, J. S. Morrow, V. T. Marchesi, and E. J. Benz. "Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species." Molecular and Cellular Biology 8, no. 1 (January 1988): 1–9. http://dx.doi.org/10.1128/mcb.8.1.1.
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The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
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Kennedy, S. P., S. L. Warren, B. G. Forget, and J. S. Morrow. "Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin." Journal of Cell Biology 115, no. 1 (October 1, 1991): 267–77. http://dx.doi.org/10.1083/jcb.115.1.267.
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Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.
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Howe, C. L., L. M. Sacramone, M. S. Mooseker, and J. S. Morrow. "Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins." Journal of Cell Biology 101, no. 4 (October 1, 1985): 1379–85. http://dx.doi.org/10.1083/jcb.101.4.1379.
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The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.
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Baines, Anthony J. "Evolution of spectrin function in cytoskeletal and membrane networks." Biochemical Society Transactions 37, no. 4 (July 22, 2009): 796–803. http://dx.doi.org/10.1042/bst0370796.
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Spectrin is a cytoskeletal protein thought to have descended from an α-actinin-like ancestor. It emerged during evolution of animals to promote integration of cells into tissues by assembling signalling and cell adhesion complexes, by enhancing the mechanical stability of membranes and by promoting assembly of specialized membrane domains. Spectrin functions as an (αβ[H])2 tetramer that cross-links transmembrane proteins, membrane lipids and the actin cytoskeleton, either directly or via adaptor proteins such as ankyrin and 4.1. In the present paper, I review recent findings on the origins and adaptations in this system. (i) The genome of the choanoflagellate Monosiga brevicollis encodes α-, β- and βHeavy-spectrin, indicating that spectrins evolved in the immediate unicellular precursors of animals. (ii) Ankyrin and 4.1 are not encoded in that genome, indicating that spectrin gained function during subsequent animal evolution. (iii) Protein 4.1 gained a spectrin-binding activity in the evolution of vertebrates. (iv) Interaction of chicken or mammal β-spectrin with PtdInsP2 can be regulated by differential mRNA splicing, which can eliminate the PH (pleckstrin homology) domain in βI- or βII-spectrins; in the case of mammalian βII-spectrin, the alternative C-terminal region encodes a phosphorylation site that regulates interaction with α-spectrin. (v) In mammalian evolution, the single pre-existing α-spectrin gene was duplicated, and one of the resulting pair (αI) neo-functionalized for rapid make-and-break of tetramers. I hypothesize that the elasticity of mammalian non-nucleated erythrocytes depends on the dynamic rearrangement of spectrin dimers/tetramers under the shearing forces experienced in circulation.
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Lawler, J., TL Coetzer, VN Mankad, RB Moore, JT Prchal, and J. Palek. "Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis." Blood 72, no. 4 (October 1, 1988): 1412–15. http://dx.doi.org/10.1182/blood.v72.4.1412.1412.
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Abstract Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.
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Lawler, J., TL Coetzer, VN Mankad, RB Moore, JT Prchal, and J. Palek. "Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis." Blood 72, no. 4 (October 1, 1988): 1412–15. http://dx.doi.org/10.1182/blood.v72.4.1412.bloodjournal7241412.
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Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.
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Coleman, T. R., A. S. Harris, S. M. Mische, M. S. Mooseker, and J. S. Morrow. "Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions." Journal of Cell Biology 104, no. 3 (March 1, 1987): 519–26. http://dx.doi.org/10.1083/jcb.104.3.519.
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The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.
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Zhou, Daixing, Jeanine A. Ursitti, and Robert J. Bloch. "Developmental Expression of Spectrins in Rat Skeletal Muscle." Molecular Biology of the Cell 9, no. 1 (January 1998): 47–61. http://dx.doi.org/10.1091/mbc.9.1.47.
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Skeletal muscle contains spectrin (or spectrin I) and fodrin (or spectrin II), members of the spectrin supergene family. We used isoform-specific antibodies and cDNA probes to investigate the molecular forms, developmental expression, and subcellular localization of the spectrins in skeletal muscle of the rat. We report that β-spectrin (βI) replaces β-fodrin (βII) at the sarcolemma as skeletal muscle fibers develop. As a result, adult muscle fibers contain only α-fodrin (αII) and the muscle isoform of β-spectrin (βIΣ2). By contrast, other types of cells present in skeletal muscle tissue, including blood vessels and nerves, contain only α- and β-fodrin. During late embryogenesis and early postnatal development, skeletal muscle fibers contain a previously unknown form of spectrin complex, consisting of α-fodrin, β-fodrin, and the muscle isoform of β-spectrin. These complexes associate with the sarcolemma to form linear membrane skeletal structures that otherwise resemble the structures found in the adult. Our results suggest that the spectrin-based membrane skeleton of muscle fibers can exist in three distinct states during development.
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Dubreuil, R. R., T. J. Byers, C. T. Stewart, and D. P. Kiehart. "A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin." Journal of Cell Biology 111, no. 5 (November 1, 1990): 1849–58. http://dx.doi.org/10.1083/jcb.111.5.1849.
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Spectrins are a major component of the membrane skeleton in many cell types where they are thought to contribute to cell form and membrane organization. Diversity among spectrin isoforms, especially their beta subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD) that we term beta H. The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit but shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family such as alpha-spectrin and alpha-actinin.
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Dubreuil, R. R., T. J. Byers, A. L. Sillman, D. Bar-Zvi, L. S. Goldstein, and D. Branton. "The complete sequence of Drosophila alpha-spectrin: conservation of structural domains between alpha-spectrins and alpha-actinin." Journal of Cell Biology 109, no. 5 (November 1, 1989): 2197–205. http://dx.doi.org/10.1083/jcb.109.5.2197.
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We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplication events.
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Hanspal, M., and J. Palek. "Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors." Journal of Cell Biology 105, no. 3 (September 1, 1987): 1417–24. http://dx.doi.org/10.1083/jcb.105.3.1417.
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The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.
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ROTTER, Björn, Yolande KROVIARSKI, Gaël NICOLAS, Didier DHERMY, and Marie-Christine LECOMTE. "alphaII-Spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding." Biochemical Journal 378, no. 1 (February 15, 2004): 161–68. http://dx.doi.org/10.1042/bj20030955.
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The spectrin–actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and µ-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to αII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves αII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that αII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.
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Lehnert, M. E., and H. F. Lodish. "Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation." Journal of Cell Biology 107, no. 2 (August 1, 1988): 413–26. http://dx.doi.org/10.1083/jcb.107.2.413.
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Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.
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Kukuła, Maciej, Beata Hanus-Lorenz, Ewa Bok, Jacek Leluk, and Aleksander F. Sikorski. "Proteins with Spectrin Motifs Which Do Not Belong to the Spectrin-α-Actinin- Dystrophin Family." Zeitschrift für Naturforschung C 59, no. 7-8 (August 1, 2004): 565–71. http://dx.doi.org/10.1515/znc-2004-7-821.
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AbstractUsing several consensus sequences for the 106 amino acid residue α-spectrin repeat segment as probes we searched animal sequence databases using the BLAST program in order to find proteins revealing limited, but significant similarity to spectrin. Among many spectrins and proteins from the spectrin-α-actinin-dystrophin family as well as sequences showing a rather high degree of similarity in very short stretches, we found seven homologous animal sequences of low overall similarity to spectrin but showing the presence of one or more spectrin-repeat motifs. The homology relationship of these sequences to α-spectrin was further analysed using the SEMIHOM program. Depending on the probe, these segments showed the presence of 6 to 26 identical amino acid residues and a variable number of semihomologous residues. Moreover, we found six protein sequences, which contained a sequence fragment sharing the SH3 (sarc homology region 3) domain homology of 42-59% similarity. Our data indicate the occurrence of motifs of significant homology to α-spectrin repeat segments among animal proteins, which are not classical members of the spectrin-α- actinin-dystrophin family. This might indicate that these segments together with the SH3 domain motif are conserved in proteins which possibly at the early stage of evolution were close cognates of spectrin-α-actinin-dystrophin progenitors but then evolved separately.
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McGough, Amy M., and Robert Josephs. "Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 510–11. http://dx.doi.org/10.1017/s0424820100122952.
Full textAbstract:
The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).
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Patel-Hett, Sunita, Hongbei Wang, Antonija J. Begonja, Jonathan N. Thon, Eva C. Alden, Nancy J. Wandersee, Xiuli An, Narla Mohandas, John H. Hartwig, and Joseph E. Italiano. "The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation." Blood 118, no. 6 (August 11, 2011): 1641–52. http://dx.doi.org/10.1182/blood-2011-01-330688.
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Abstract Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer–disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the “barbell shapes” of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.
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Deng, H., J. K. Lee, L. S. Goldstein, and D. Branton. "Drosophila development requires spectrin network formation." Journal of Cell Biology 128, no. 1 (January 1, 1995): 71–79. http://dx.doi.org/10.1083/jcb.128.1.71.
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The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.
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Byers, T. J., R. Dubreuil, D. Branton, D. P. Kiehart, and L. S. Goldstein. "Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins." Journal of Cell Biology 105, no. 5 (November 1, 1987): 2103–10. http://dx.doi.org/10.1083/jcb.105.5.2103.
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Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.
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Schneider, A., H. U. Lutz, R. Marugg, P. Gehr, and T. Seebeck. "Spectrin-like proteins in the paraflagellar rod structure of Trypanosoma brucei." Journal of Cell Science 90, no. 2 (June 1, 1988): 307–15. http://dx.doi.org/10.1242/jcs.90.2.307.
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A polyclonal, monospecific rabbit antibody to human erythrocyte spectrins cross-reacted with two sets of proteins (a doublet of 180/200K and a triplet of 67–66-65K; K = 10(3) Mr) in the parasitic protozoon Trypanosoma brucei brucei. Except for the 66K protein, the cross-reacting proteins are localized in the flagellum, on the basis of evidence from cell fractionation and immunofluorescence microscopy. Immunogold labelling and electron micrographs further revealed that the spectrin-like proteins are confined to the paraflagellar rod structure. The spectrin-like proteins with apparent molecular weights of 180 and 200 share homology with spectrin band 1, since V8-protease from Staphylococcus aureus generated similarly sized, antigenic peptides from these proteins. The results indicate homology between the cross-reacting proteins and human red cell spectrin.
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Thomas, G. H., and J. A. Williams. "Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila." Journal of Cell Science 112, no. 17 (September 1, 1999): 2843–52. http://dx.doi.org/10.1242/jcs.112.17.2843.
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The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known (beta)-spectrin isoforms: a conventional (beta)-spectrin chain, and the novel isoform (beta)(Heavy)-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two (beta)-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing (beta)(Heavy)-spectrin into which the basolateral (beta)-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of (beta)-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of (beta)(Heavy)-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms.
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Park, Sunghyouk, Michael E. Johnson, and Leslie W. M. Fung. "Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin." Blood 100, no. 1 (July 1, 2002): 283–88. http://dx.doi.org/10.1182/blood.v100.1.283.
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Abstract Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the N-terminal region of α-spectrin, with the Arg28 position considered to be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Spα1-156, consisting of the first 156 residues in the N-terminal region of α-spectrin (αN). The association of these α-spectrin peptides that have single amino acid replacements with a β-spectrin model peptide, consisting of the C-terminal region of β-spectrin (βC), was determined, and structural changes due to amino acid replacements were monitored by nuclear magnetic resonance (NMR). We found evidence for similar and very localized structural changes in Spα1-156Arg45Thr and Spα1-156Arg45Ser, although these 2 mutant peptides associated with β-spectrin peptide with significantly differing affinities. The Spα1-156Arg28Ser peptide showed an affinity for the β-spectrin peptide comparable to that of Spα1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that both Arg45 replacements induce only minor structural perturbations in the first helix of Spα1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results also indicate that the mechanism for reduced spectrin tetramerization is through mutation-induced changes in molecular recognition at the αβ-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.
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Frappier, T., F. Stetzkowski-Marden, and L. A. Pradel. "Interaction domains of neurofilament light chain and brain spectrin." Biochemical Journal 275, no. 2 (April 15, 1991): 521–27. http://dx.doi.org/10.1042/bj2750521.
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We have previously demonstrated that brain spectrin binds to the low-molecular-mass subunit of neurofilaments (NF-L) [Frappier, Regnouf & Pradel (1987) Eur. J. Biochem. 169, 651-657]. In the present study, we seek to locate their respective binding domains. In the first part we demonstrate that brain spectrin binds to a 20 kDa domain of NF-L. This domain is part of the rod domain of neurofilaments and plays a role in the polymerization process. However, the polymerization state does not seem to have any influence on the interaction. In the second part, we provide evidence that NF-L binds to the beta-subunit of not only brain spectrin but also human and avian erythrocyte spectrins. The microtubule-associated protein, MAP2, which has also been shown to bind to microfilaments and neurofilaments, binds to the same domain of NF-L as spectrin does. Finally, among the tryptic peptides of brain spectrin, we show that some peptides of low molecular mass (35, 25, 20 and 18 kDa) co-sediment with either NF-L or F-actin.
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Dubreuil, R., T. J. Byers, D. Branton, L. S. Goldstein, and D. P. Kiehart. "Drosophilia spectrin. I. Characterization of the purified protein." Journal of Cell Biology 105, no. 5 (November 1, 1987): 2095–102. http://dx.doi.org/10.1083/jcb.105.5.2095.
Full textAbstract:
We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.
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Stabach, Paul R., Ivana Simonović, Miranda A. Ranieri, Michael S. Aboodi, Thomas A. Steitz, Miljan Simonović, and Jon S. Morrow. "The structure of the ankyrin-binding site of β-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties." Blood 113, no. 22 (May 28, 2009): 5377–84. http://dx.doi.org/10.1182/blood-2008-10-184291.
Full textAbstract:
Spectrin and ankyrin participate in membrane organization, stability, signal transduction, and protein targeting; their interaction is critical for erythrocyte stability. Repeats 14 and 15 of βI-spectrin are crucial for ankyrin recognition, yet the way spectrin binds ankyrin while preserving its repeat structure is unknown. We have solved the crystal structure of the βI-spectrin 14,15 di-repeat unit to 2.1 Å resolution and found 14 residues critical for ankyrin binding that map to the end of the helix C of repeat 14, the linker region, and the B-C loop of repeat 15. The tilt (64°) across the 14,15 linker is greater than in any published di-repeat structure, suggesting that the relative positioning of the two repeats is important for ankyrin binding. We propose that a lack of structural constraints on linker and inter-helix loops allows proteins containing spectrin-like di-repeats to evolve diverse but specific ligand-recognition sites without compromising the structure of the repeat unit. The linker regions between repeats are thus critical determinants of both spectrin's flexibility and polyfunctionality. The putative coupling of flexibility and ligand binding suggests a mechanism by which spectrin might participate in mechanosensory regulation.
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Malchiodi-Albedi, F., M. Ceccarini, J. C. Winkelmann, J. S. Morrow, and T. C. Petrucci. "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." Journal of Cell Science 106, no. 1 (September 1, 1993): 67–78. http://dx.doi.org/10.1242/jcs.106.1.67.
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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)
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Evans, SS, WC Wang, CC Gregorio, T. Han, and EA Repasky. "Interferon-alpha alters spectrin organization in normal and leukemic human B lymphocytes." Blood 81, no. 3 (February 1, 1993): 759–66. http://dx.doi.org/10.1182/blood.v81.3.759.bloodjournal813759.
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Interferon-alpha (IFN-alpha) regulates the growth, differentiation, and recirculation of normal and malignant B lymphocytes. In this report we examine the effects of IFN-alpha on the distribution of the cytoskeletal protein spectrin in peripheral blood B lymphocytes from normal donors and patients diagnosed with chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). Exposure of normal and leukemic B cells to IFN-alpha in vitro was shown by immunofluorescence microscopy to cause a dose-dependent increase in the percentage of cells containing discrete focal accumulations of spectrin, ie, a single large aggregate or cap-like structure near the plasma membrane. Although the magnitude of this effect was variable among individual patient samples, in some experiments IFN-alpha induced a fourfold increase in the percentage of leukemic B cells exhibiting focal accumulations of spectrin. Spectrin reorganization induced by IFN-alpha was abrogated by the protein synthesis inhibitor cycloheximide. In addition, IFN-alpha increased the total cellular content of spectrin in B-CLL cells by approximately twofold to fourfold. Finally, a role for protein kinase C in mediating the effects of IFN-alpha on spectrin's organization is implicated by studies in which calphostin C inhibited the IFN-induced focal accumulation of spectrin. Taken together, these studies suggest that the immunomodulatory activities of IFN-alpha in normal and malignant B cells involve a change in the organization of the spectrin- based cytoskeleton.
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Evans, SS, WC Wang, CC Gregorio, T. Han, and EA Repasky. "Interferon-alpha alters spectrin organization in normal and leukemic human B lymphocytes." Blood 81, no. 3 (February 1, 1993): 759–66. http://dx.doi.org/10.1182/blood.v81.3.759.759.
Full textAbstract:
Abstract Interferon-alpha (IFN-alpha) regulates the growth, differentiation, and recirculation of normal and malignant B lymphocytes. In this report we examine the effects of IFN-alpha on the distribution of the cytoskeletal protein spectrin in peripheral blood B lymphocytes from normal donors and patients diagnosed with chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). Exposure of normal and leukemic B cells to IFN-alpha in vitro was shown by immunofluorescence microscopy to cause a dose-dependent increase in the percentage of cells containing discrete focal accumulations of spectrin, ie, a single large aggregate or cap-like structure near the plasma membrane. Although the magnitude of this effect was variable among individual patient samples, in some experiments IFN-alpha induced a fourfold increase in the percentage of leukemic B cells exhibiting focal accumulations of spectrin. Spectrin reorganization induced by IFN-alpha was abrogated by the protein synthesis inhibitor cycloheximide. In addition, IFN-alpha increased the total cellular content of spectrin in B-CLL cells by approximately twofold to fourfold. Finally, a role for protein kinase C in mediating the effects of IFN-alpha on spectrin's organization is implicated by studies in which calphostin C inhibited the IFN-induced focal accumulation of spectrin. Taken together, these studies suggest that the immunomodulatory activities of IFN-alpha in normal and malignant B cells involve a change in the organization of the spectrin- based cytoskeleton.
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Lambert, Muriel W. "Spectrin and its interacting partners in nuclear structure and function." Experimental Biology and Medicine 243, no. 6 (March 2018): 507–24. http://dx.doi.org/10.1177/1535370218763563.
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Nonerythroid αII-spectrin is a structural protein whose roles in the nucleus have just begun to be explored. αII-spectrin is an important component of the nucleoskelelton and has both structural and non-structural functions. Its best known role is in repair of DNA ICLs both in genomic and telomeric DNA. αII-spectrin aids in the recruitment of repair proteins to sites of damage and a proposed mechanism of action is presented. It interacts with a number of different groups of proteins in the nucleus, indicating it has roles in additional cellular functions. αII-spectrin, in its structural role, associates/co-purifies with proteins important in maintaining the architecture and mechanical properties of the nucleus such as lamin, emerin, actin, protein 4.1, nuclear myosin, and SUN proteins. It is important for the resilience and elasticity of the nucleus. Thus, αII-spectrin’s role in cellular functions is complex due to its structural as well as non-structural roles and understanding the consequences of a loss or deficiency of αII-spectrin in the nucleus is a significant challenge. In the bone marrow failure disorder, Fanconi anemia, there is a deficiency in αII-spectrin and, among other characteristics, there is defective DNA repair, chromosome instability, and congenital abnormalities. One may speculate that a deficiency in αII-spectrin plays an important role not only in the DNA repair defect but also in the congenital anomalies observed in Fanconi anemia , particularly since αII-spectrin has been shown to be important in embryonic development in a mouse model. The dual roles of αII-spectrin in the nucleus in both structural and non-structural functions make this an extremely important protein which needs to be investigated further. Such investigations should help unravel the complexities of αII-spectrin’s interactions with other nuclear proteins and enhance our understanding of the pathogenesis of disorders, such as Fanconi anemia , in which there is a deficiency in αII-spectrin. Impact statement The nucleoskeleton is critical for maintaining the architecture and functional integrity of the nucleus. Nonerythroid α-spectrin (αIISp) is an essential nucleoskeletal protein; however, its interactions with other structural and non-structural nuclear proteins and its functional importance in the nucleus have only begun to be explored. This review addresses these issues. It describes αIISp’s association with DNA repair proteins and at least one proposed mechanism of action for its role in DNA repair. Specific interactions of αIISp with other nucleoskeletal proteins as well as its important role in the biomechanical properties of the nucleus are reviewed. The consequences of loss of αIISp, in disorders such as Fanconi anemia, are examined, providing insights into the profound impact of this loss on critical processes known to be abnormal in FA, such as development, carcinogenesis, cancer progression and cellular functions dependent upon αIISp’s interactions with other nucleoskeletal proteins.
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Wolgast, Lucia R., Linda A. Cannizzarro, K. H. Ramesh, Xiaonan Xue, Dan Wang, Pritish K. Bhattacharyya, Jerald Z. Gong, et al. "Spectrin Isoforms." American Journal of Clinical Pathology 136, no. 2 (August 2011): 300–308. http://dx.doi.org/10.1309/ajcpsa5rnm9igfjf.
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Franck, Paul Hubert Frans, Cobie Postma, Marjan Veuger, Pierre Wijermans, and Frans A. Kuypers. "A Family with Hereditary Elliptocytosis: Variable Clinical Severity Caused by Three Mutations in the α-Spectrin Gene,." Blood 118, no. 21 (November 18, 2011): 3167. http://dx.doi.org/10.1182/blood.v118.21.3167.3167.
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Abstract Abstract 3167 Introduction The membrane of erythrocytes is composed of a bilayer of phospholipids and cholesterol. It is strengthened by a membraneskeleton consisting of the proteins spectrin, ankyrin, pallidin, band 3 and band 4.1. Hereditary elliptocytosis (HE) is caused by mutations in the spectrin protein, resulting in a typical elliptocytic shape. These cells have a decreased deformability and a shortened lifespan. Most mutations in HE are located in the head to head self association site of the α- and β dimers of spectrin. HE Patients with heterozygous mutations in α spectrin show little or no hemolysis because α spectrin is synthesized in an excess relative to β spectrin. In the heterozygous situation, plenty of normal wild type α spectrine (Wt α) is available for incorporation in to the membraneskeleton. In Hereditary Pyropoikilocytosis (HPP) with its bizarre shapes, a second mutation in the α spectrin is present, in addition to the HE mutation. It is responsible for a defect in the synthesis of α spectrin, resulting in the production of 50% functional α spectrin. This mutation is called LELY (Low Expression LYon). Thus, when the LELY mutation is located in trans on the allele in respect to the HE allele, the expression of the Wt α spectrin protein is reduced. As a consequence, more abnormal HE spectrin will be incorporated in to the membraneskeleton. This enhanced expression of the HE mutation results in an unstable membraneskeleton of the HPP cell. Method The deformability of erythrocytes is measured using the ektacytometer LORRCA Maxsis of Mechatronics (Hoorn, The Netherlands). DNA was isolated from white blood cells from peripheral blood. The HE mutations are found by a PCR of the α spectrin exons where most mutations exists for HE, followed by DNA sequencing using the ABI prism genetic analyzer from Applied Biosystems. The LELY mutation is proven by a PCR of exon 40 of the α spectrin gene followed by RFLP agarose gel electrophoresis.(59 G/A) mutation and the LELY mutation are present. In addition to these Exon 2 and LELY mutations, a third mutation in Exon 6 (103 T/C) is found in the α spectrin of brother E.α spectrin is synthesized and incorporated into the membraneskeleton. The same mutations hold for brother E., but he has HPP. This is attributed to a third additional mutation in Exon 6 of the α spectrin gene. This Exon 6 mutation is located in trans to the Exon 2 mutation that in turn is in cis with the LELY mutation. Like in brother W. the expression of the Exon 2 mutated spectrin is reduced due to the LELY mutation. However relatively more of the Exon 6 mutated α spectrin comes available for the dimerization with β-spectrin resulting in an unstable membrane skeleton, causing the HPP of brother E. Results The B. family is examined for the presence of HE or HPP (figure 1). Brother E. has clinical symptoms, poikilocytes and a typical ektacytometric deformability matching HPP. Brother W. and his daughter N. have no clinical symptoms but elliptocytes and an ektacytometric deformability typical for HE. In all affected individuals the same Exon 2. Conclusion The combination of Exon 2 and LELY mutations normally leads to HPP. Concerning brother W. and his daughter N. this is not the case. They have a mild form of HE. The explanation for this finding is, that the Exon 2- and LELY mutation are located in a cis rather than in a trans position on the α spectrin gene (figure 1). In that case the mutated Exon 2 is less expressed and more Wt. Disclosures: No relevant conflicts of interest to declare.
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Yang, Yang, Yasuhiro Ogawa, Kristian L. Hedstrom, and Matthew N. Rasband. "βIV spectrin is recruited to axon initial segments and nodes of Ranvier by ankyrinG." Journal of Cell Biology 176, no. 4 (February 5, 2007): 509–19. http://dx.doi.org/10.1083/jcb.200610128.
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High densities of ion channels at axon initial segments (AISs) and nodes of Ranvier are required for initiation, propagation, and modulation of action potentials in axons. The organization of these membrane domains depends on a specialized cytoskeleton consisting of two submembranous cytoskeletal and scaffolding proteins, ankyrinG (ankG) and βIV spectrin. However, it is not known which of these proteins is the principal organizer, or if the mechanisms governing formation of the cytoskeleton at the AIS also apply to nodes. We identify a distinct protein domain in βIV spectrin required for its localization to the AIS, and show that this domain mediates βIV spectrin's interaction with ankG. Dominant-negative ankG disrupts βIV spectrin localization, but does not alter endogenous ankG or Na+ channel clustering at the AIS. Finally, using adenovirus for transgene delivery into myelinated neurons, we demonstrate that βIV spectrin recruitment to nodes of Ranvier also depends on binding to ankG.
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Lorenzo, Damaris N., Alexandra Badea, Ruobo Zhou, Peter J. Mohler, Xiaowei Zhuang, and Vann Bennett. "βII-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport." Proceedings of the National Academy of Sciences 116, no. 31 (June 17, 2019): 15686–95. http://dx.doi.org/10.1073/pnas.1820649116.
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βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. βII-spectrin–null neurons exhibited reduced axon growth, loss of actin–spectrin-based periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that βII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. βII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a βII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of βII-spectrin and AnkB nearly eliminated transport. Thus, βII-spectrin promotes both axon growth and axon stability through establishing the actin–spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo.
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Matsushita, Hirokazu, Matthew Vesely, Ravindra Uppaluri, and Robert Schreiber. "Antigen immunoselection as a mechanism of cancer immunoediting (165.3)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 165.3. http://dx.doi.org/10.4049/jimmunol.186.supp.165.3.
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Abstract Cancer immunoediting is the process wherein the immune system not only protects against tumor development but also promotes outgrowth of tumors with reduced immunogenicity. Although we and others identified several key immune components that participate in this process, we know very little about the targets of cancer immunoediting. In this study we used a highly immunogenic methylcholanthrene induced sarcoma cell line (d42m1) to ask whether tumor antigens can be immunoediting targets. Like most unedited MCA sarcomas, d42m1 cells form tumors when transplanted into immunodeficient mice but are rejected in naive syngeneic wild type (WT) mice. We identified two antigens of d42m1 by expression cloning: a point mutant of spectrin-β2 and a point mutant of M-phase phosphoprotein 8 (MPP8). In WT mice, d42m1 occasionally produces escape variants lacking mutant spectrin-β2 but maintaining expression of mutant MPP8 that can form tumors in naive WT mice. Enforced expression of mutant but not WT spectin-β2 into escape variant cell lines converted them into regressors. Analysis of the parental d42m1 cell line revealed that only 80% of d42m1 clones expressed mutant spectrin-β2 whereas 100% expressed mutant MPP8. Only spectrin-β2 expressing d42m1 clones were rejected in WT mice. Thus, mutant spectrin-β2 is not only the major rejection antigen of d42m1 sarcoma cells but is the target of a cancer immunoediting immunoselection process that facilitates tumor escape from immune control.
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Berghs, Stanny, Diego Aggujaro, Ronald Dirkx, Elena Maksimova, Paul Stabach, Jean-Michel Hermel, Jian-Ping Zhang, et al. "βiv Spectrin, a New Spectrin Localized at Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System." Journal of Cell Biology 151, no. 5 (November 27, 2000): 985–1002. http://dx.doi.org/10.1083/jcb.151.5.985.
Full textAbstract:
We report the identification of βIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The βIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of βIV spectrin generates at least four distinct isoforms, numbered βIVΣ1–βIVΣ4 spectrin. The longest isoform (βIVΣ1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. βIVΣ2 and βIVΣ3 spectrin encompass the NH2- and COOH-terminal halves of βIVΣ1 spectrin, respectively, while βIVΣ4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of βIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, βIVΣ1 spectrin is recognized as a protein of 250 kD. Anti–βIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from βIVΣ1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for βIV spectrin is more prominent in α than in β cells. In brain, βIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrinG 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, βIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, βIVΣ1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that βIVΣ1 spectrin interacts with ankyrinG 480/270-kD and participates in the clustering of voltage-gated Na+ channels and cell-adhesion molecules at initial segments and nodes of Ranvier.
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Higgs, Henry N. "Spectres of spectrin: molecular modeling and hemolytic disease." Trends in Biochemical Sciences 26, no. 12 (December 2001): 702. http://dx.doi.org/10.1016/s0968-0004(01)02004-7.
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Hanspal, M., JS Hanspal, KE Sahr, E. Fibach, J. Nachman, and J. Palek. "Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis." Blood 82, no. 5 (September 1, 1993): 1652–60. http://dx.doi.org/10.1182/blood.v82.5.1652.1652.
Full textAbstract:
Abstract Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.
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Hanspal, M., JS Hanspal, KE Sahr, E. Fibach, J. Nachman, and J. Palek. "Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis." Blood 82, no. 5 (September 1, 1993): 1652–60. http://dx.doi.org/10.1182/blood.v82.5.1652.bloodjournal8251652.
Full textAbstract:
Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.
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Leshchyns'ka, Iryna, Vladimir Sytnyk, Jon S. Morrow, and Melitta Schachner. "Neural cell adhesion molecule (NCAM) association with PKCβ2 via βI spectrin is implicated in NCAM-mediated neurite outgrowth." Journal of Cell Biology 161, no. 3 (May 12, 2003): 625–39. http://dx.doi.org/10.1083/jcb.200303020.
Full textAbstract:
In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100–insoluble complexes with βI spectrin. Heteromeric spectrin (αIβI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the βI spectrin fragment encompassing second and third spectrin repeats (βI2–3). In NCAM120-transfected cells, βI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-β-cyclodextrin disrupts the NCAM120–spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180–βI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCβ2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180–spectrin–PKCβ2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative βI2–3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCβ2 and NCAM140 or NCAM180. Redistribution of PKCβ2 to NCAM–spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with βI2–3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM–spectrin–PKCβ2 complex is necessary for NCAM-mediated neurite outgrowth.
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Mazock, G. Harper, Amlan Das, Christine Base, and Ronald R. Dubreuil. "Transgene Rescue Identifies an Essential Function for Drosophila β Spectrin in the Nervous System and a Selective Requirement for Ankyrin-2–binding Activity." Molecular Biology of the Cell 21, no. 16 (August 15, 2010): 2860–68. http://dx.doi.org/10.1091/mbc.e10-03-0180.
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The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential β spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a β spectrin transgene or to knock down β spectrin expression with dsRNA. The results show that 1) overexpression of β spectrin in most of the cell types studied was lethal; 2) knockdown of β spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-β spectrin transgene was sufficient to overcome the lethality of a loss-of-function β spectrin mutation. Thus β spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for β spectrin function in vivo.
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Hulsmeier, J., J. Pielage, C. Rickert, G. M. Technau, C. Klambt, and T. Stork. "Distinct functions of -Spectrin and -Spectrin during axonal pathfinding." Development 134, no. 4 (January 10, 2007): 713–22. http://dx.doi.org/10.1242/dev.02758.
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Coetzer, T., J. Lawler, JT Prchal, and J. Palek. "Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis." Blood 70, no. 3 (September 1, 1987): 766–72. http://dx.doi.org/10.1182/blood.v70.3.766.766.
Full textAbstract:
Abstract The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.
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Coetzer, T., J. Lawler, JT Prchal, and J. Palek. "Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis." Blood 70, no. 3 (September 1, 1987): 766–72. http://dx.doi.org/10.1182/blood.v70.3.766.bloodjournal703766.
Full textAbstract:
The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.
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Li, Donghai. "Role of Spectrin in Endocytosis." Cells 11, no. 15 (August 8, 2022): 2459. http://dx.doi.org/10.3390/cells11152459.
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Cytoskeletal spectrin is found in (non)erythroid cells. Eukaryotic endocytosis takes place for internalizing cargos from extracellular milieu. The role of spectrin in endocytosis still remains poorly understood. Here, I summarize current knowledge of spectrin function, spectrin-based cytoskeleton and endocytosis of erythrocytes, and highlight how spectrin contributes to endocytosis and working models in different types of cells. From an evolutionary viewpoint, I discuss spectrin and endocytosis in a range of organisms, particularly in plants and yeast where spectrin is absent. Together, the role of spectrin in endocytosis is related to its post-translational modification, movement/rearrangement, elimination (by proteases) and meshwork fencing.
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Cohen, AM, SC Liu, LH Derick, and J. Palek. "Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes." Blood 68, no. 4 (October 1, 1986): 920–26. http://dx.doi.org/10.1182/blood.v68.4.920.920.
Full textAbstract:
Abstract 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.
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Cohen, AM, SC Liu, LH Derick, and J. Palek. "Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes." Blood 68, no. 4 (October 1, 1986): 920–26. http://dx.doi.org/10.1182/blood.v68.4.920.bloodjournal684920.
Full textAbstract:
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.