RESUMEN
Formation of the neuromuscular junction requires the release of agrin from the presynaptic terminal of motor neurons. Clustering of acetylcholine receptors (AChRs) on the postsynaptic sarcolemma is initiated by agrin-dependent activation of the muscle-specific kinase. While the postsynaptic scaffolding protein rapsyn is vital for high density AChR aggregation, little is known about the mechanism through which AChRs are immobilized on the postsynaptic membrane. Ultrastructural and immunohistochemical studies of rat skeletal muscle have suggested that AChRs are anchored to a membrane-associated cytoskeleton that contains spectrin-like proteins and is thus similar to that of the human erythrocyte [Bloch RJ, Bezakova G, Ursitti JA, Zhou D, Pumplin DW (1997) A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle. Soc Gen Physiol Ser 52:177-195]. We are studying a protein of the spectrin superfamily, ACF7 (also known as MACF), as a postsynaptic cytoskeletal component of the neuromuscular junction. ACF7 has multiple cytoskeleton-binding domains, including an N-terminal actin-binding domain that, we postulate, may interact with rapsyn, the scaffolding protein that binds directly to AChRs. To test this hypothesis, we co-expressed fragments of these molecules in cultured fibroblasts and assessed their co-distribution and interaction using confocal microscopy and co-immunoprecipitation. We demonstrate that the actin-binding domain of ACF7 specifically interacts with the tetratricopeptide repeat domains of rapsyn. Furthermore, we show using surface plasmon resonance and blot overlay that the actin-binding domain of ACF7 binds directly to rapsyn. These results suggest that, in mammalian skeletal muscle, AChRs are immobilized in the membrane through rapsyn-mediated anchoring to an ACF7-containing network that in turn is linked to the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación/métodos , Ratones , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Mutagénesis/fisiología , Mioblastos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Ratas , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie/métodos , Transfección/métodosRESUMEN
We studied the spectrins in developing hippocampal tissue in vivo and in vitro to learn how they contribute to the organization of synaptic and extrasynaptic regions of the neuronal plasma membrane. beta-Spectrin, but not beta-fodrin or alpha-fodrin, increased substantially during postnatal development in the hippocampus, where it was localized in neurons but not in astrocytes. Immunoprecipitations from neonatal and adult hippocampal extracts suggest that while both beta-spectrin and beta-fodrin form heteromers with alpha-fodrin, oligomers containing all three subunits are also present. At the subcellular level, beta-fodrin and alpha-fodrin were present in the cell bodies, dendrites, and axons of pyramidal-like neurons in culture, as well as in astrocytes. beta-Spectrin, by contrast, was absent from axons but present in cell bodies and dendrites, where it was organized in a loose, membrane-associated meshwork that lacked alpha-fodrin. A similar meshwork was also apparent in pyramidal neurons in vivo. At some dendritic spines, alpha-fodrin was present in the necks but not in the heads, whereas beta-spectrin was present at significant levels in the spine heads. The presence of significant amounts of beta-spectrin without an accompanying alpha-fodrin subunit was confirmed by immunoprecipitations from extracts of adult hippocampus. Our results suggest that the spectrins in hippocampal neurons can assemble to form different membrane-associated structures in distinct membrane domains, including those at synapses.
Asunto(s)
Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Neuronas/química , Espectrina/análisis , Factores de Edad , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos , Axones/química , Proteínas Portadoras/análisis , Proteínas Portadoras/inmunología , Membrana Celular/química , Células Cultivadas , Dendritas/química , Inmunohistoquímica , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/inmunología , Neuronas/ultraestructura , Ratas , Espectrina/inmunología , Sinapsis/químicaRESUMEN
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.
Asunto(s)
Músculo Esquelético/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrina/metabolismo , Animales , Ancirinas/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente , Pruebas de Precipitina , Conejos , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/enzimologíaRESUMEN
This unit contains procedures for electrophoretically transferring proteins onto a variety of membranes including polyvinylidene difluoride (PVDF) and nitrocellulose, and derivatized membranes. The choice of membrane type for electrotransfer is dependent on the ultimate application for the blot membrane. An alternate protocol is provided for electroblotting in semidry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents.
Asunto(s)
Resinas Acrílicas , Western Blotting/métodos , Electroforesis/métodos , Colodión , Detergentes , Membranas Artificiales , Compuestos Orgánicos , Polivinilos , Proteínas/química , Análisis de Secuencia de Proteína , SolventesRESUMEN
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 beta-spectrin (betaI) replaces beta-fodrin (betaII) at the sarcolemma as skeletal muscle fibers develop. As a result, adult muscle fibers contain only alpha-fodrin (alphaII) and the muscle isoform of beta-spectrin (betaISigma2). By contrast, other types of cells present in skeletal muscle tissue, including blood vessels and nerves, contain only alpha- and beta-fodrin. During late embryogenesis and early postnatal development, skeletal muscle fibers contain a previously unknown form of spectrin complex, consisting of alpha-fodrin, beta-fodrin, and the muscle isoform of beta-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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/metabolismo , Espectrina/genética , Animales , Animales Recién Nacidos , Northern Blotting , Femenino , Desarrollo de Músculos , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Pruebas de Precipitina , Ratas , Espectrina/inmunología , Espectrina/metabolismoRESUMEN
Human erythroid alpha-spectrin alleles responsible for hereditary elliptocytosis (alphaHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism alphaLELY (LELY: Low Expression LYon) occurs in trans. The alphaLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA --> GTA, Leu --> Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the alpha-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of alpha-chains from the alphaLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif alpha subunit peptides containing the dimer initiation region. The two forms of alpha spectrin produced by alternative mRNA splicing of the alphaLELY allele were represented by alpha18-21(1857), a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and alpha18-21(1857-Delta46), a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with alpha18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the alphaIV and alphaV domains that was the characteristic feature initially used to identify the alphaLELY (SpalphaV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the alpha21 motif, because this region of the alpha18-21(1857-Delta46) peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an alphaLELY homozygote and the two alphaLELY recombinant peptides strongly suggests that little, if any, of the 50% of the alpha chains from the alphaLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant alphaLELY peptides, the inability of detectable amounts of exon 46(-) alpha chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.
Asunto(s)
Eritrocitos/metabolismo , Exones , Espectrina/química , Espectrina/genética , Alelos , Dimerización , Humanos , Empalme del ARNRESUMEN
We have presented ultrastructural and semiquantitative immunofluorescence evidence to support the idea that AChR are clustered in rat myotubes by virtue of their ability to associate with a spectrin-based membrane skeleton. Many of the interactions postulated to be involved in the formation of this skeleton, and in the anchoring of AChR to it, must still be examined at the biochemical level, but the overall similarity of this structure to that of the human erythrocyte is already clear. The ability of different members of the spectrin superfamily to associate in various combinations to form distinct plasmalemmal domains provides some exciting hints as to how the surface membrane can be organized efficiently to subserve multiple purposes. One of the challenges of future research will be to learn how innervation regulates the assembly of the membrane skeleton at the developing NMJ, and how this structure is altered as the junction matures. Another will be to learn if the principles of neuromuscular synaptogenesis are relevant to interactions between neurons in the brain, where cells must distinguish between multiple synaptic inputs and assemble synaptic structures at thousands of distinct sites on the neurolemma. Members of the spectrin superfamily have been identified in synaptic structures in the central nervous system (e.g., Carlin et al., 1983; LeVine and Sahyoun, 1986; Malchiodi-Albedi et al., 1993), so much of what we have learned at the neuromuscular junction may be applicable to central synapses.
Asunto(s)
Citoesqueleto/química , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Citoesqueleto/metabolismo , Humanos , Músculo Esquelético/química , Músculo Esquelético/ultraestructura , Unión Neuromuscular/química , Unión Neuromuscular/metabolismoRESUMEN
Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N- and C-terminal truncations of one or more motifs from beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nM, while each additional motif added only 0.85 nM to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.
Asunto(s)
Actinina/metabolismo , Eritrocitos/metabolismo , Espectrina/genética , Actinina/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Biopolímeros , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrina/química , Espectrina/metabolismoRESUMEN
The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33 +/- 5 nm) at the central junctions are proposed to be stabilized and limited by association with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropomodulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropomodulin and tropomyosin are associated with the erythrocyte actin filaments in situ.
Asunto(s)
Actinas/análisis , Proteínas Portadoras/análisis , Citoesqueleto/química , Eritrocitos/química , Proteínas de Microfilamentos , Tropomiosina/análisis , Citoesqueleto/ultraestructura , Electroforesis en Gel de Poliacrilamida , Eritrocitos/ultraestructura , Grabado por Congelación , Humanos , Inmunohistoquímica , Microscopía Inmunoelectrónica , Coloración Negativa , TropomodulinaRESUMEN
The crystal structure of the three-helix-bundle spectrin motif provides new insights into the structure of the large proteins that constitute the spectrin superfamily, which includes dystrophin and alpha-actinin.
Asunto(s)
Actinina/química , Distrofina/química , Conformación Proteica , Estructura Secundaria de Proteína , Espectrina/química , Animales , Cristalografía por Rayos X , Drosophila , Membrana Eritrocítica/ultraestructura , Humanos , Sustancias Macromoleculares , Modelos MolecularesRESUMEN
Head-to-head association of two spectrin alpha beta heterodimers to form tetramers involves the formation of two equivalent alpha-beta complexes. The sites on the alpha subunit N-terminal region and beta subunit C-terminal region that form these alpha beta complexes have been identified using protease footprinting and direct binding assays. The existence of a similar previously hypothesized internal head-to-head alpha beta interaction in dimers was also demonstrated. The discrete regions of both subunits that are protected from proteolysis in tetramers and dimers are not due to the laterally associated subunit since head-to-head complexes of a univalent alpha peptide with a univalent beta peptide show similar protection of the same sites. These sites are unshielded immediately after monomers assemble side-to-side to form heterodimers, demonstrating that reconstituted dimers are initially in an "open" conformation. Conversion of open dimers to a closed form through formation of the internal head-to-head alpha beta association, as demonstrated by restoration of protease protection, occurred on a time scale of hours at 0 degrees C. Analysis of peptide binding affinities as well as isolation and sequence analysis of head-to-head alpha beta noncovalent complexes further defined the regions required for association on both subunits. These regions are homologous to the 106-residue repetitive motif that comprises most of both chains. An algorithm designed to improve prediction accuracy of multiple homologous motifs was used to model the conformation of spectrin repetitive motifs as well as the contact regions. In this model, the separate alpha and beta binding sites are incomplete complementary parts of a triple stranded folding unit. Formation of the alpha beta head-to-head complex produces a triple stranded conformational unit that is slightly different from other homologous motifs in the protein. Most hemolytic anemia mutations that are known to disrupt tetramer association are located in the mapped regions, including several mutations that induce a conformational change in the paired subunit.
Asunto(s)
Eritrocitos/metabolismo , Espectrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Humanos , Hidrólisis , Datos de Secuencia Molecular , Conformación Proteica , Espectrina/químicaRESUMEN
Isolated skeletons from human erythrocyte ghosts were studied using immunogold labeling; negative staining; and quick-freeze, deep-etch, rotary replication with Pt/C (QFDERR). Isolated skeletons visualized by QFDERR were similar to the negatively stained skeletons in that the proteins spectrin, actin, and ankyrin could be easily distinguished. However, the quick-frozen skeletons had two fewer filaments (4.2 +/- 0.7) at an actin junction. Immunogold labeling of skeletons with site-specific spectrin antibodies not only confirmed the designation of these filaments as spectrin molecules, but indicated that about 30% of spectrin filaments form non-actin junctions consistent with the hexameric organization of these filaments. Many of the filaments displayed a striking banding pattern indicative of underlying substructure. Isolated skeletons prepared by QFDERR also showed evidence of laterally associated spectrin filaments. These associations, as well as many hexamer junctions, are lost during negative staining. Negative staining also apparently caused approximately 21% of the spectrin filaments to separate into their monomeric subunits. These results indicate that the surface tension imposed during negative staining of isolated skeletons can cause a loss of interactions normally present in the intact membrane skeleton.
Asunto(s)
Membrana Eritrocítica/ultraestructura , Membrana Eritrocítica/química , Grabado por Congelación , Humanos , Inmunohistoquímica , Coloración Negativa , Platino (Metal) , Técnicas de Réplica , Técnica Histológica de SombreadoRESUMEN
We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by approximately 3 nm.
Asunto(s)
Citoesqueleto/ultraestructura , Eritrocitos/ultraestructura , Citoesqueleto de Actina/ultraestructura , Proteína 1 de Intercambio de Anión de Eritrocito/ultraestructura , Membrana Eritrocítica/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica , Técnicas de Réplica , Manejo de Especímenes , Espectrina/ultraestructuraRESUMEN
We have made several technical improvements for quick-freeze, deep-etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly-L-lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick-freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (less than 10 microns). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table--one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.