Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.     

Mutation in exon 1f of PLEC, leading to disruption of plectin isoform 1f, causes autosomal-recessive limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy (LGMD) is a genetically heterogeneous group of inherited muscular disorders manifesting symmetric, proximal, and slowly progressive muscle weakness. Using Affymetrix 250K SNP Array genotyping and homozygosity mapping, we mapped an autosomal-recessive LGMD phenotype to the telomeric portion of chromosome 8q in a consanguineous Turkish family with three affected individuals. DNA sequence analysis of PLEC identified a homozygous c.1_9del mutation containing an initiation codon in exon 1f, which is an isoform-specific sequence of plectin isoform 1f. The same homozygous mutation was also detected in two additional families during the analysis of 72 independent LGMD2-affected families. Moreover, we showed that the expression of PLEC was reduced in the patient's muscle and that there was almost no expression for plectin 1f mRNA as a result of the mutation. In addition to dystrophic changes in muscle, ultrastructural alterations, such as membrane duplications, an enlarged space between the membrane and sarcomere, and misalignment of Z-disks, were observed by transmission electron microscopy. Unlike the control skeletal muscle, no sarcolemmal staining of plectin was detected in the patient's muscle. We conclude that as a result of plectin 1f deficiency, the linkage between the sarcolemma and sarcomere is broken, which could affect the structural organization of the myofiber. Our data show that one of the isoforms of plectin plays a key role in skeletal muscle function and that disruption of the plectin 1f can cause the LGMD2 phenotype without any dermatologic component as was previously reported with mutations in constant exons of PLEC.     

Primary longitudinal adhesion structures: plectin-containing precursors of costameres in differentiating human skeletal muscle cells

Plectin is a high molecular mass protein (ca 530 kDa) that binds actin, intermediate filaments, and microtubules. Mutations of the human plectin gene cause epidermolysis bullosa simplex with muscular dystrophy. In mature human skeletal muscle, plectin is localized between neighboring myofibrils and between myofibrils and the sarcolemma, both at the level of Z-discs. In the present study we have analyzed plectin expression patterns with emphasis on its sarcolemmal localization during human skeletal muscle differentiation in vitro. In myoblasts plectin showed a cytoplasmic intermediate filament-like distribution, whereas in myotubes plectin is also found at the level of the sarcolemma. In particular, in early myotubes a specific plectin isoform colocalizes with the costameric proteins vinculin and beta1D integrin in longitudinally orientated structures which increased in number and longitudinal extension upon further maturation. In mature myotubes processes perpendicular to the parallel system of longitudinal structures became apparent. Subsequent to the occurrence of spontaneous myofibrillar contractions, the number of longitudinal streaks decreased, and plectin and other costameric proteins were found in an orderly cross-striated sarcolemmal lattice overlying myofibrillar Z-discs. Our study demonstrates that plectin is preassembled together with vinculin and beta1D integrin into primary longitudinal adhesion structures. After the occurrence of spontaneous contractions, these structures reorient and mature costameres are assembled.     

Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle.

Plectin is a multifunctional cytoskeletal linker protein with an intermediate filament-binding site and sequence elements with high homology to actin-binding domains. Mutations of the human plectin gene as well as the targeted inactivation of its murine analog cause a generalized blistering skin disorder and muscular dystrophy, thus implying its essential role in cells that are exposed to mechanical stress. In the present study we report the characterization of two new domain-specific plectin antibodies as well as ultrastructural localization of plectin in normal human skeletal muscle. Using immunogold electron microscopy, we localized plectin at three prominent sites: 1) Plectin is found at regularly spaced intervals along the cytoplasmic face of the plasma membrane. 2) It is distinctly localized at filamentous bridges between Z-lines of peripheral myofibrils and the sarcolemma and 3) at structures forming the intermyofibrillar scaffold. At the latter two locations, plectin and desmin were found to colocalize. Our ultrastructural analysis suggests that plectin may have a central role in the structural and functional organization of the intermediate filament cytoskeleton in mature human skeletal muscle.     

BPAG1 isoform-b: Complex distribution pattern in striated and heart muscle and association with plectin and α-actinin

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein α-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like α-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and α-actinin-2, and its NH₂-terminus, which contains an actin-binding domain, directly interacted with that of plectin and α-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.     

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.     

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.     

Novel interactions of ankyrins-G at the costameres: the muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein–protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein–protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres and sarcolemma.     

Plectin–intermediate filament partnership in skin, skeletal muscle, and peripheral nerve

Plectin is a large, 500-kDa, intermediate filament (IF)-associated protein. It acts as a cytoskeletal crosslinker and signaling scaffold, affecting mechanical as well as dynamic properties of the cytoskeleton. As a member of the plakin family of cytolinker proteins, plectin has a multidomain structure that is responsible for its vast binding portfolio. It not only binds to all types of IFs, actin filaments and microtubules, but also to transmembrane receptors, proteins of the subplasma membrane protein skeleton, components of the nuclear envelope, and several kinases with known roles in migration, proliferation, and energy metabolism of cells. Due to alternative splicing, plectin is expressed as various isoforms with differing N-terminal heads that dictate their differential subcellular targeting. Through specific interactions with other proteins at their target sites and their ability to bind to all types of IFs, plectin molecules provide strategically located IF anchorage sites within the cytoplasm of cells. In this review, we will present an overview of the structural features and functional properties of plectin and discuss recent progress in defining the role of its isoforms in stress-prone tissues and the implicated diseases, with focus on skin, skeletal muscle, and Schwann cells of peripheral nerve.     

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.     

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.     

Association of Mitochondria with Plectin and Desmin Intermediate Filaments in Striated Muscle

Plectin (M(r) > 500,000) is a versatile and widely expressed cytolinker protein. In striated muscle it is predominantly found at the Z-disc level where it colocalizes with the intermediate filament protein desmin. Both proteins show altered labeling patterns in tissues of muscular dystrophy patients. Moreover, mutations in the plectin gene lead to the autosomal recessive human disorder epidermolysis bullosa simplex with muscular dystrophy, and defects in the desmin gene have been shown to cause familiar cardiac and skeletal myopathy. Since intermediate filaments (IFs) in striated muscle tissue have been found to be intimately associated with mitochondria, we investigated whether plectin is involved in this association. Using postembedding immunogold labeling of Lowicryl sections and immunogold labeling of ultrathin cryosections, we show that plectin is associated with desmin IFs linking myofibrils to mitochondria at the level of the Z-disc and along the entire length of the sarcomere. The localization of plectin label at the mitochondrial membrane itself was consistent with a putative linker function of plectin between desmin IFs and the mitochondrial surface. In mitochondrion-rich muscle fibers, both plectin and desmin were part of an ordered arrangement of mitochondrial side branches, which wound around myofibrils adjacent to the Z-discs and were anchored into a filamentous network transversing from one fibril to the other. The association of mitochondria with plectin and IFs was seen also in tissues without regular distribution patterns of mitochondria, such as heart muscle and neonatal skeletal muscle tissues. These data were supplemented with in vitro binding assays showing direct interaction of plectin with desmin via its carboxy-terminal IF-binding domain. As a cytolinker protein associated with mitochondria and desmin IFs, plectin could play an important role in the positioning and shape formation, in particular branching, of mitochondrial organelles in striated muscle tissues.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

Four and a half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.     

Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma

mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.     

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 domainspecific 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.     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Aquaporin-4 water channel oligomers are associated with the transverse tubules of skeletal myofibers

Transverse (T) tubules comprise a tortuous network inside the skeletal myofibers enclosing a distinct osmotic environment. Here we have examined whether the T tubules contain aquaporin type 4 (AQP4) water channels to mediate rapid transmembrane water flow. Separation of T tubular and sarcolemmal membranes by sucrose density gradient centrifugation revealed that two main isoforms of AQP4, namely M23 and M1, were present in both membrane fractions. Compatible with this, expression of fluorescent Venus-AQP4.M23 in rat muscle showed the protein both in the T tubules and at the sarcolemma. Blue-Native polyacrylamide gel electrophoresis showed that higher order oligomers typical to the AQP4 water channel were present in both membrane compartments. Interestingly, α-syntrophin that mediates binding of AQP4 to the sarcolemmal dystrophin glycoprotein complex was also present in the T tubule fraction. Deletion of the syntrophin-binding sequence of AQP4 increased its mobile fraction at the sarcolemma but not in the T tubules. Taken together, our results strongly suggest that both the sarcolemma and the T tubules harbor higher order oligomers of the AQP4 water channel but the interactions with adjacent macromolecules are different.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.