Spectrin-based Membrane Skeleton Research Articles

Release of an ∼55 kDa fragment containing the actin-binding domain of β-spectrin by caspase-8 during FND-induced apoptosis depends on the presence of protein 4.1

Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of β-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ∼55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ∼55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (<30–120min) submembranous colocalization of both proteins. Proteolytic release of the N-terminal ∼55kDa fragment of purified spectrin by recombinant caspase-8 does not occur in normal cells, but does occur in isolated membrane, such as red blood cell ghosts, or in vitro in the presence of apoptotic cell extracts. Surprisingly, proteolysis of purified spectrin by recombinant caspase-8 resulted in the generation of the ∼55kDa fragment only in the presence of purified protein 4.1. This suggests that only the appropriate spatial arrangement of the spectrin-based membrane skeleton or the appropriate conformational state of spectrin, which are both known to be induced by 4.1, can sensitize β-spectrin to cleavage by caspase-8 at the N-terminal ABD-containing region.

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation

AbstractMegakaryocytes 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.

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.

Site-Directed Spin-Labeling Studies: Structure of the AnkD34-Cdb3 Complex

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton thereby stabilizing the membrane. Atomic resolution structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a twelve ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER), have been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top convex surface of AnkD34 along the flexible linker away from the ankyrin groove primarily through hydrophobic interactions. This is a previously structurally uncharacterized surface for binding of cdb3 to AnkD34. Since a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to Ankyrin-R in the erythrocyte membrane. Supported by NIH P01 GM080513.

YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the Kd values for V22F were similar to those for the wild-type (about 7 nM), whereas the Kd values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.

Dematin, a Component of the Erythrocyte Membrane Skeleton, Is Internalized by the Malaria Parasite and Associates with Plasmodium 14-3-3

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.

On the association of lipid rafts to the spectrin skeleton in human erythrocytes

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule.

Evolution of the spectrin-based membrane skeleton

Evolution of the spectrin-based membrane skeleton

Evolution of the spectrin-based membrane skeleton

A group of four proteins – spectrin, ankyrin, 4.1 and adducin – evolved with the metazoa. These membrane-cytoskeletal proteins cross-link actin on the cytoplasmic face of plasma membranes and link a variety of transmembrane proteins to the cytoskeleton. In this paper, the evolution of these proteins is analysed. Genomics indicate that spectrin was the first to appear, since the genome of the choanoflagellate Monosiga brevicolis contains genes for α, β and βH spectrin. This organism represents a lineage of free-living and colonial protists from which the metazoa are considered to have diverged. This indicates that spectrin emerged in evolution before the animals. Simple animals such as the placozoan Trichoplax adherens also contain recognizable precursors of 4.1, ankyrin and adducin, but these could probably not bind spectrin. Ankyrin and adducin seem to have acquired spectrin-binding activity with the appearance of tissues since they appear to have largely the same domain structure in all eumetazoa. 4.1 was adapted more recently, with the emergence of the vertebrates, to bind spectrin and promote its interaction with actin. A simple hypothesis is that spectrin was prerequisite (but not sufficient) for animal life; that spectrin interaction with ankyrin and adducin was required for evolution of major tissues; and that 4.1 acquired a spectrin-actin binding activity as animal size increased with the appearance of vertebrates. The spectrin/ankyrin/adducin/4.1 complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Na,K-ATPase in skeletal muscle: two populations of β-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules

We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules.

Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

AbstractThe erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: Cross-reaction with proteins other than spectrin

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane’s structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight αβ-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti αβ-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.

Tmod3 regulates polarized epithelial cell morphology

Although the role of the actin cytoskeleton in morphogenesis of polarized epithelial sheets is generally accepted as centrally important, the regulation of actin dynamics in this process remains unclear. Here, we show that the pointed-end capping protein Tmod3 contributes to epithelial cell shape within confluent monolayers of polarized epithelial cells. Tmod3 localizes to lateral cell membranes in polarized epithelia of several cell types. Reduction of Tmod3 levels by shRNA leads to a loss of F-actin and tropomyosins from lateral cell membranes, and a decrease in epithelial cell height, without effects on localisation of tight junction or adherens junction proteins, or any apparent changes in cell-cell adhesion. Instead, distribution of alphaII-spectrin on lateral membranes is disrupted upon reduction of Tmod3 levels, suggesting that loss of Tmod3 function leads to destabilization and disassembly of tropomyosin-coated actin filaments followed by disorganization of the spectrin-based membrane skeleton on lateral membranes. These data demonstrate for the first time a role for pointed-end capping in morphology regulation of polarized epithelial cells through stabilization of F-actin on lateral membranes. We propose that Tmod3-capped tropomyosin-actin filaments provide crucial links in the spectrin membrane skeleton of polarized epithelial cells, enabling the membrane skeleton to maintain cell shape.

A Mutant αII-spectrin Designed to Resist Calpain and Caspase Cleavage Questions the Functional Importance of This Process in Vivo

alpha- and beta-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous alphaII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of alphaII-spectrin by these enzymes could be involved in important functions. To assess the role of alphaII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant alphaII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant alphaII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular alphaII-spectrin cleavage in either major cellular functions or in normal development.

New insights into function of red cell membrane proteins and their interaction with spectrin-based membrane skeleton

New insights into function of red cell membrane proteins and their interaction with spectrin-based membrane skeleton

The N-terminal 11 amino acids of human erythrocyte band 3 are critical for aldolase binding and protein phosphorylation: implications for band 3 function

The 911 amino acid band 3 (SLC4A1) is the major intrinsic membrane protein of red cells and is the principal Cl-/HCO3- exchanger. The N-terminal cytoplasmic domain of band 3 anchors the spectrin-based membrane skeleton to the lipid bilayer through its interaction with ankyrin and also binds glycolytic enzymes and hemoglobin. We identified a son of a consanguineous marriage with severe anemia in association with marked deficiency of band 3 (12% +/- 4% of normal). Direct nucleotide sequencing of SLC4A1 gene demonstrated a single base substitution (T --> C) at position + 2 in the donor splice site of intron 2, resulting in the generation of a novel mutant protein. Biochemical characterization of the mutant protein showed that it lacked the first 11 N-terminal amino acids (band 3 Neapolis). The expression of the mutant protein resulted in the complete absence of membrane-bound aldolase, and the mutant band 3 could not be tyrosine phosphorylated. The ability of the malarial parasite P falciparum to invade these red cells was significantly decreased. The identification of a novel band 3 mutant and its structural and functional characterization enabled us to identify pivotal roles for the 11 N-terminal amino acids in several protein functions and, in turn, in red-cell physiology.