Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.     

Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.     

Absence of utrophin in intercalated discs of human cardiac muscle

Utrophin is the autosomal homologue of dystrophin. In normal skeletal muscle it is localised only to neuromuscular and myotendinous junctions, nerves and vascular tissue. In Xp21 muscular dystrophies, utrophin is also detected on the sarcolemma of skeletal and cardiac muscle, while dystrophin is absent or reduced. In normal cardiac muscle, some reports have demonstrated utrophin at intercalated discs and T-tubules. We have re-examined the distribution of utrophin in normal human cardiac muscle using a panel of eight monoclonal antibodies against different epitopes in N- and C-terminal domains. In contrast to previous studies, utrophin was not detected at the intercalated discs or T-tubules, although labelling of blood vessels was strong. We conclude that the primary location of utrophin in normal heart is in the vascular system. In addition, our results show that the utrophin on cardiac blood vessels is full length, similar to that of skeletal muscle blood vessels.     

Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: distribution and localization in muscles

We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms.     

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.     

Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice.

Desmin, the main component of intermediate filaments (IFs) in mature skeletal muscle, forms an interlinking scaffold around myofibrils with connections to the sarcolemma and the nuclear membrane. Desmin is enriched in neuromuscular and myotendinous junctions. Mice lacking the desmin gene develop normally and reproduce. However, postnatally they develop a cardiomyopathy and a dystrophy in highly used muscles. We have investigated whether and how neuromuscular and myotendinous junctions are affected and whether nestin compensates for the lack of desmin in the knock-out (K/O) mice. We show that neither neuromuscular nor myotendinous junctions were markedly affected in the desmin K/O mice. In neuromuscular junctions nestin was present between the postjunctional folds and the subneural nuclei and between the nucleus and the myofibrillar cytoskeleton. In myotendinous junctions nestin was present between myofibrils at the Z-disc level and in longitudinal strands close to and at the junction. Nestin expression at these specialized sites, as well as during myogenesis and myofibrillogenesis, is independent of the presence of desmin. In desmin K/O mice nestin was also found in regenerating myofibers. The presence of nestin at neuromuscular and myotendinous junctions might provide enough strength for preservation and organization of the junctional areas, although desmin is lacking.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

Occurrence and immunolocalization of plectin in tissues

Various tissues from rat were examined for the occurrence and cellular localization of plectin, a 300,000-dalton polypeptide component present in intermediate filament-enriched cytoskeletons prepared from cultured cells by treatment with nonionic detergent and high salt solution. The extraction of liver, heart, skeletal muscle, tongue, and urinary bladder with 1% Triton/0.6 M KCl yielded insoluble cell residues that contained polypeptides of Mr 300,000 in variable amounts. These high Mr polypeptide species and a few bands of slightly lower Mr (most likely proteolytic breakdown products) were shown to react with antibodies to rat glioma C6 cell plectin using immunoautoradiography and/or immunoprecipitation. By indirect immunofluorescence microscopy using frozen sections (4 micron) of stomach, kidney, small intestine, liver, uterus, urinary bladder, and heart, antigens reacting with antibodies to plectin were found in fibroblast, endothelial, smooth, skeletal, and cardiac muscle, nerve, and epithelial cells of various types. Depending on the cell type, staining was observed either throughout the cytoplasm, or primarily at the periphery of cells, or in both locations. In hepatocytes, besides granular staining at the cell periphery, conspicuous staining of junctions sealing bile canaliculi was seen. In cardiac muscle strong staining was seen at intercalated disks and, as in skeletal muscle, at Z-lines. In cross sections through smooth muscle, most strikingly of urinary bladder, antibodies to plectin specifically decorated regularly spaced, spot-like structures at the cell periphery. By immunoelectron microscopy using the peroxidase technique, antiplectin-reactive material was found along cell junctions of hepatocytes and was particularly enriched at desmosomal plaques and structures associated with their cytoplasmic surfaces. A specific immunoreaction with desmosomes was also evident in sections through tongue. In cardiac muscle, besides Z-lines, intercalated disks were reactive along almost their entire surface, suggesting that plectin was associated with the fascia adherens, desmosomes, and probably gap junctions. In smooth muscle cells, regularly spaced lateral densities probably representing myofilament attachment sites were immunoreactive with plectin antibodies. The results show that plectin is of widespread occurrence with regard to tissues and cell types. Furthermore, immunolocalization by light and electron microscopy at junctional sites of various cell types and at attachment sites of cytoplasmic filaments in epithelial and muscle cells suggests that plectin possibly plays a universal role in the formation of cell junctions and the anchorage of cytoplasmic filaments.     

Formation of myoneural and myotendinous junctions in the chick embryo

Summary The mode of formation of the myoneural and myotendinous junctions was investigated in the thigh muscles of the chick embryo. Myotendinous junctions first appeared on day 11 of incubation, whereas myoneural junctions developed on day 12. Intracellular AChE activity in the muscles increased by the 12th day of incubation, and decreased rapidly after the formation of the myoneural junctions. Light and electron microscopically, AChE activity was demonstrated in the nuclear envelope, sarcoplasmic reticulum, Golgi complex, and in large granules which appeared to be derived from the Golgi complex. Large granules showing an intense AChE activity accumulated in the sarcoplasm at the poles of the muscle fiber before the formation of myotendinous junctions. After the translocation of this intracellular enzyme onto the sarcolemma, most likely the result of an exocytosis of the granules, the myotendinous junctions were formed. The AChE-rich granules present in the middle of myotubes developed into spindle- or comma-shaped cisternae which were located in the sarcoplasm just below the presumptive motor endplates. The present results suggest that the transport of AChE-rich granules to the sarcolemma is the first step in the formation of myoneural and myotendinous junctions.This work was carried out under grant 38848 from the Ministry of Education of Japan     

Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

Biochemical and immunohistochemical evidence for a non-muscle myosin at the neuromuscular junction in bovine skeletal muscle.

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac beta-myosin heavy chain (beta-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac beta-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac beta-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.     

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.     

Talin at myotendinous junctions

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.     

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.     

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

Isolation and localization of filamin in heart muscle

High-molecular-mass protein was isolated from chicken heart muscle. The apparent molecular mass of a single polypeptide chain is similar to that of chicken gizzard filamin: 250-270 kDa. The protein interacts with antibodies against chicken gizzard filamin and induces F-actin gelation in a concentration-dependent manner. Immunofluorescent staining of cardiomyocytes and chicken heart sections with antifilamin antibody demonstrates two types of filamin localization: filamin was located on the sarcomere border in the periphery of the Z-disk; filamin was found in intercalated disks between cardiomyocytes.     

Alpha-actinin is absent from the terminal segments of myofibrils and from subsarcolemmal densities in frog skeletal muscle

The presence and distribution of alpha-actinin, an actin-bundling protein, was investigated at sites where frog skeletal muscle forms junctions with tendon collagen fibers. These sites, called myotendinous junctions, are regions where myofibrils terminate and where the force of muscular contraction is transmitted from muscle cells to the substratum. An antibody manufactured to chicken smooth muscle alpha-actinin was used as a probe for alpha-actinin localization in this study. The cross-reactivity of this antibody with frog skeletal muscle alpha-actinin is demonstrated in immunoblots of one-dimensional (1D) electrophoretic separations of muscle proteins. Immunofluorescent localization of anti-alpha-actinin and electron microscopic immunolabelling confirms that the antibody binds to Z-discs with high affinity. However, in sections treated for electron microscopy with affinity-purified anti-alpha-actinin and a ferritin-conjugated, second antibody, there was no significant difference between experimental or control preparations in the number of ferritin grains overlying dense, subsarcolemmal material at junctional or non-junctional regions. Furthermore, Z-discs near myotendinous junctions displayed less binding of anti-alpha-actinin than Z-discs located several micrometers or more from the cells' termini. These findings indicate that thin filaments are not bundled by alpha-actinin near the sarcolemma. The results also provide evidence for molecular heterogeneity between Z-discs at the ends of muscle cells compared with other regions of the cell in that the terminal Z-discs of myofibrils contain very little or no alpha-actinin relative to non-terminal Z-discs.     

Immunolocalization of ubiquitin conjugates at Z-bands and intercalated discs of rat cardiomyocytes in vitro and in vivo.

Ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells, can be covalently bound to a wide variety of proteins in the nucleus, cytosol, cytoskeleton, and plasmalemma. This diversity of target proteins reflects a diversity of functions for ubiquitin conjugation. Previous studies have showed enhanced localization of ubiquitin conjugates to Z-bands of normal skeletal muscle and increased ubiquitination in atrophic muscles. These results have implicated a ubiquitin-mediated pathway in protein turnover and degradation in striated muscle. To investigate whether such a pathway might also exist in cardiac striated muscle, we used an affinity-purified polyclonal antibody (conjugate specific) and indirect immunofluorescence to localize ubiquitin conjugates in neonatal and adult rat cardiac myocytes both in vitro and in vivo. In both cultured myocytes and heart tissue, fluorescent ubiquitin conjugates were found in the nucleus as aggregates, in the cytoplasm in a striated pattern indicative of Z-bands, and in intercellular junctions at the intercalated discs between myocytes. Although the acceptor proteins and the physiological significance of ubiquitination at these locations are unknown, the targeting of ubiquitin to specific sites within the nucleus, myofibrils, and sarcolemma could provide a means for selective processing of individual components within these larger macromolecular assemblies, thus implying a regulatory role for ubiquitin conjugation in turnover or stability of proteins in the heart.     

A novel marker of tissue junctions, collagen XXII

Here we describe a novel specific component of tissue junctions, collagen XXII. It was first identified by screening an EST data base and subsequently expressed as a recombinant protein and characterized as an authentic tissue component. The COL22A1 gene on human chromosome 8q24.2 encodes a collagen that structurally belongs to the FACIT protein family (fibril-associated collagens with interrupted triple helices). Collagen XXII exhibits a striking restricted localization at tissue junctions such as the myotendinous junction in skeletal and heart muscle, the articular cartilage-synovial fluid junction, or the border between the anagen hair follicle and the dermis in the skin. It is deposited in the basement membrane zone of the myotendinous junction and the hair follicle and associated with the extrafibrillar matrix in cartilage. In situ hybridization of myotendinous junctions revealed that muscle cells produce collagen XXII, and functional tests demonstrated that collagen XXII acts as a cell adhesion ligand for skin epithelial cells and fibroblasts. This novel gene product, collagen XXII, is the first specific extracellular matrix protein present only at tissue junctions.