Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice.

We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.

The actin binding domain of ACF7 binds directly to the tetratricopeptide repeat domains of rapsyn.

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.

The tetratricopeptide repeat domains of rapsyn bind directly to cytoplasmic sequences of the muscle-specific kinase.

Clustering of acetylcholine receptors at the developing vertebrate neuromuscular junction is initiated by neural agrin, which stimulates the activity of the muscle-specific kinase (MuSK). Acetylcholine receptor clustering is also dependent on the postsynaptic scaffolding protein, rapsyn, which binds to acetylcholine receptors. Here, we address the possibility that MuSK and rapsyn bind directly to each other by coexpressing sequences of the cytoplasmic domain of MuSK with rapsyn in COS-7 cells and assaying for codistribution and biochemical interaction. Sequences constituting the bulk of the kinase domain can interact with rapsyn. This interaction is mediated by the tetratricopeptide repeat domains, but not the coiled coil or zinc finger domains, of rapsyn. This interaction does not require tyrosine phosphorylation of the MuSK sequences. Binding is direct, as indicated by blot overlay and surface plasmon resonance experiments. The sequence of the cytoplasmic domain of MuSK that most effectively codistributes with rapsyn confers the ability of an otherwise inactive receptor tyrosine kinase, TrkA, to associate with rapsyn. Our results support a model in which the tetratricopeptide repeat domains of rapsyn bind directly to the cytoplasmic portion of MuSK, which could thereby serve as an initial scaffold for the clustering of acetylcholine receptors.

Spectrins in developing rat hippocampal cells.

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

Na,K-ATPase in skeletal muscle: two populations of beta-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.

Monocarboxylic acid transporters, MCT1 and MCT2, in cortical astrocytes in vitro and in vivo.

We used sequence-specific antibodies to characterize two monocarboxylic acid transporters, MCT1 and MCT2, in astrocytes. Both proteins are expressed in primary cultures of cortical astrocytes, as indicated by immunoblotting and immunofluorescence. Both MCT1 and MCT2 are present in small, punctate structures in the cytoplasm and at the cell membrane. Cells showing very low levels of labeling for glial fibrillary acidic protein (GFAP) also label more dimly for MCT2, but not for MCT1. In vivo, double-label immunofluorescence studies coupled with confocal microscopy indicate that MCT1 and MCT2 are rare in astrocytes in the cortex. However, they are specifically labeled in astrocytes of the glial limiting membrane and in white matter tracts. Both transporters are also present in the microvasculature. Comparison of labeling for MCT1 and MCT2 with markers of the blood-brain barrier shows that the transporters are not always limited to the astrocytic endfeet in vivo. Our results suggest that the level of expression of monocarboxylic acid transporters MCT1 and MCT2 by cortical astrocytes in vivo is significantly lower than in vitro but that astrocytes in some other regions of the brain can express one or both proteins in significant amounts.

Membrane skeleton of innervated and denervated fast- and slow-twitch muscle.

We used confocal microscopy and immunoblotting to study membrane skeletal proteins of fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of the adult rat. In the extensor digitorum longus (EDL), beta-spectrin concentrates in costameres, whereas dystrophin is enriched at costameres but is also present in intercostameric regions. In the soleus, beta-spectrin and dystrophin underlie much of the sarcolemma, and intercostameric regions are difficult to detect. The EDL sarcolemma reorganizes following denervation to resemble soleus sarcolemma, but denervation does not significantly affect the latter. Consistent with these observations, soleus contains similar amounts of dystrophin but more beta-spectrin than EDL. Denervation increases beta-spectrin levels only in the EDL and dystrophin levels in both muscles. Denervation does not affect beta-fodrin, a beta-spectrin homolog expressed in embryonic myofibers. Thus, neuromuscular activity controls sarcolemmal organization and the levels of beta-spectrin and dystrophin, but not postnatal downregulation of beta-fodrin. The differences in organization of the sarcolemma may underlie the differential susceptibility of fast and slow myofibers to dystrophinopathies.

Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes.

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.

Differential distribution of dystrophin and beta-spectrin at the sarcolemma of fast twitch skeletal muscle fibers.

We used double label immunofluorescence and confocal microscopy to examine the organization of beta-spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both beta-spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable beta-spectrin. The dystrophin-associated proteins, syntrophin and beta-dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without beta-spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on beta-spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.

Extensive but coordinated reorganization of the membrane skeleton in myofibers of dystrophic (mdx) mice.

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. beta-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, beta-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, beta-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain beta-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with beta-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including alpha-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.

The zinc finger domain of the 43-kDa receptor-associated protein, rapsyn: role in acetylcholine receptor clustering.

We injected rat myotubes with proteins and antibodies to assess the importance of the zinc finger (ZnF) domain of the 43-kDa receptor-associated protein, rapsyn, in clustering acetylcholine receptors (AChR). Injection of rat myotubes with a fusion protein containing the ZnF domain of rapsyn disrupted AChR clusters. Clusters were unaffected by a fusion protein containing a double mutant that does not bind zinc. Similar results were obtained with the purified wild type and mutant ZnF domains. The ZnF of HIV-1 nucleocapsid protein had no effect. AChR clusters were also disrupted in myotubes injected with antibodies to the ZnF domain, followed by injection of anti-antibodies. Injection of antibodies directed against a different rapsyn epitope or against the cytoplasmic domain of the AChR had no effect. In transfection experiments with HEK 293 cells, the ZnF domain failed to associate with membrane aggregates containing full-length rapsyn, AChR, or rapsyn and AChR together. We conclude that the ZnF domain of rapsyn provides a binding site essential for AChR clustering, but that this site is unlikely to be involved in high affinity binding of rapsyn to itself or to AChR.

The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its beta5 subunit.

Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing beta-3 and beta-5integrins) and fibronectin receptors (FnR, containing beta1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the beta5 subunit of the VnR at both domains. We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the beta5 subunit of the VnR. Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human beta5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous beta1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the beta5 but not the beta1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the beta5 but not the beta1 peptide. Thus, a sequence including the NPXY motif in the integrin beta5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.

Developmental expression of spectrins in rat skeletal muscle.

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.

Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle.

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.

Two populations of beta-spectrin in rat skeletal muscle.

We use immunoblotting, immunoprecipitation, and centrifugation in sucrose density gradients to show that the product of the erythrocyte beta-spectrin gene in rat skeletal muscle (muscle beta-spectrin) is present in two states, one associated with fodrin, and another that is not associated with any identifiable spectrin or fodrin subunit. Immunofluorescence studies indicate that a significant amount of beta-spectrin without alpha-fodrin is present in the myoplasm of some muscle fibers, and, more strikingly, at distinct regions of the sarcolemma. These results suggest that alpha-fodrin and muscle beta-spectrin associate in muscle in situ, but that some muscle beta-spectrin without a paired alpha-subunit forms distinct domains at the sarcolemma.

Cytoskeleton-membrane interactions at the postsynaptic density of Xenopus neuromuscular junctions.

Acetylcholine receptors of mature muscle fibres are concentrated in the postsynaptic membrane by mechanisms that are not yet understood. As one possibility, receptors might be anchored to cytoskeletal elements in the postsynaptic density that is located beneath the membrane where receptors are concentrated. To address this possibility, we examined the cytoskeleton at the postsynaptic density and determined the organization of cytoskeletal filaments relative to clustered acetylcholine receptors (AChR). Xenopus nerve-muscle co-cultures were sheared to expose the cytoplasmic membrane surface, then quick-frozen, deep-etched, and rotary-replicated. Areas with a high concentration of AChR had aggregates of particles protruding from the cytoplasmic surface of the membrane, with actin microfilaments attached to and cross-linking the aggregates. Microfilaments contacted only a few of the particles in an aggregate. These findings suggest that short-range interactions may bind individual AChR into small aggregates, while microfilaments tie these aggregates together at the nerve-muscle junction.

Microfilament-membrane interactions in Xenopus myocytes.

We used quick-freeze, deep-etch, rotary-replication transmission electron microscopy to determine at molecular resolution the organization of microfilaments at the cytoplasmic surface of the sarcolemma of Xenopus myocytes. We demonstrate that actin microfilaments interact with the sarcolemma in two distinct ways. In one, which resembled focal contacts in Xenopus fibroblasts [Samuelsson et al., 1993: J. Cell Biol. 122:485-496], bundles of microfilaments approached the sarcolemma at sites containing aggregates of membrane-associated particles. Immunogold cytochemistry showed that these particle aggregates contained vinculin, talin and beta 1-integrin. In the second, which covered most of the cytoplasmic surface of the sarcolemma, individual actin microfilaments formed an extensive, lattice-like array. Particle aggregates associated with this array of actin microfilaments also labeled with antibodies to vinculin, talin and beta 1-integrin. The unique, lattice-like association of actin microfilaments with the membrane in Xenopus myocytes suggests that the organization of actin filaments over most of the sarcolemma is distinct from focal contacts, mediating widespread associations of the actin cytoskeleton with the cytoplasmic membrane face.

Synapse development. Up the junction.

alpha-Dystroglycan has been suggested to be the receptor for agrin, an extracellular glycoprotein that signals postsynaptic differentiation at the neuromuscular junction, but it may not have the necessary specificity.