Correspondence to: Siegfried Labeit, Klinikum Mannheim, Theodor-Kutzer-Ufer-1, Mannheim 68167, Germany. Tel:(49) 621-383-2422 Fax:(49) 621-383-1971 E-mail:labeit{at}embl-heidelberg.de.
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Abstract |
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We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of -actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and
-actininbinding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH2-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH2-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladinCARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via
-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).
Key Words:
-actinin, nebulin, palladin, myopalladin, CARP
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Introduction |
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The precise organization of Z-lines, the borders of individual sarcomeres in vertebrate striated muscle, is a remarkable example of supramolecular assembly in eukaryotic cells. Z-lines contain the barbed ends of actin thin filaments, the NH2-terminal ends of titin filaments, the COOH-terminal ends of nebulin filaments (skeletal muscle), and nebulette (cardiac muscle), as well as a variety of other regulatory and structural proteins. In addition to being a boundary between successive sarcomeres, Z-lines are responsible for transmitting tension generated by individual sarcomeres along the length of the myofibril, allowing for efficient contractile activity (for discussion see
Detailed ultrastructural and biochemical investigations of the Z-disc and its various components have yielded valuable information concerning its structural architecture. The width of the Z-line can vary from 30 nm in fish skeletal muscle, up to >1 µm in patients with certain forms of nemaline myopathy (
-actinin (
-actininbinding sites within titin's NH2-terminal, 80-kD, Z-disc integral segment. These binding sites (the Z-repeats and titin's NH2-terminal sequences adjacent to the Ig-repeat Z4), may link together titin and
-actinin filaments both inside the Z-line and at its periphery (
50-kD COOH-terminal region of nebulin also extends into the Z-line lattice in skeletal muscle: the Src homology (SH)1 3 domain of nebulin is localized
25 nm inside the Z-line, whereas the more NH2-terminal repeating modules of nebulin are located at the periphery of the Z-line (
To date, the molecular interactions that are responsible for anchoring nebulin and nebulette filaments inside Z-lines are unknown, although previous models have proposed potential direct interactions between nebulin/nebulette and -actinin (
-actinin's EF hand region. Our in vitro binding data predict that myopalladin tethers the nebulin (directly), titin, and thin filaments (indirectly) via
-actinin filaments, thereby forming intraZ-line meshworks. Additionally, we found that the NH2-terminal region of myopalladin interacts with the cardiac ankyrin repeat protein (CARP) (
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Materials and Methods |
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Yeast Two-Hybrid Interaction Studies
For a survey of potential nebulin and myopalladin interactions, cDNA fragments were amplified from rabbit psoas and human skeletal muscle cDNA by PCR (5 d). In screens where the pAS2-1 bait vector was used, the plates were supplemented with 1,5 mM 3-amino-1,2,4-triazole (3-AT; Sigma-Aldrich).
Transformants were picked, restreaked onto SD/Leu-/Trp-/His-plates, and screened for ß-galactosidase activity. ß-Galactosidase activity of the cells was measured either by colony lift filter assays using X-gal or in liquid culture using chlorophenol red-ß-D-galactopyranoside (cPRG) as described by the manufacturer (CLONTECH Laboratories, Inc.). ß-Galactosidasepositive colonies were processed to loosen the bait plasmid, and prey clones were recovered by electroporation of yeast DNA in Escherichia coli and sequenced. To further confirm binding, transformants were retransformed into yeast with either the bait or the vector. In addition, the inserts of the bait and the prey vector were swapped and cotransformed into yeast.
For -actininmyopalladin interaction studies, a previously described
actinin-2 deletion series in the pGAD424 vector was used (
actinin-3 containing two 4-EF hands cloned in pGAD10 was used (provided by Alan Beggs, Children's Hospital, Boston, MA). The sequence of all constructs was verified by sequencing.
cDNA Cloning and Sequence Analysis
The identified myopalladin prey cDNA corresponded to a 780-bp partial cDNA clone. The partial myopalladin cDNA was labeled randomly with 32P (Optiprime kit; Stratagene) and hybridized to a human heart cDNA library (number 936208; Stratagene). From a total of 400,000 screened clones, 80 clones hybridized to the probe. 24 myopalladin-positive phages were randomly picked and characterized by PCR, using combinations of specific internal primers and vector armderived primers. Clones extending 12 kb into the 5' and 3' directions were selected for sequencing. In total, three fragments extending towards the 5' end, and one fragment extending towards the 3' end provided a 5,696-bp cDNA. The presence of 5' and 3' untranslated regions, a putative start ATG at the 5' end, and polyadenylation at the 3' end indicated that the 5.7 kb cDNA corresponded to the complete myopalladin message (see Fig 4 A). The complete myopalladin coding sequence was also amplified from a human skeletal muscle cDNA library by PCR (HL4010AB; CLONTECH Laboratories, Inc.) and sequenced. No sequence differences between cardiac and skeletal myopalladin were observed. All myopalladin fragments were sequenced on both DNA strands. Reads were edited and assembled using Geneskipper v1.2 software (Christian Schwager, European Molecular Biology Laboratory). For sequence analysis, Ig-I repeats were aligned using ClustalX (
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Protein Expression and Antibody Production
For obtaining specific antibodies against myopalladin and palladin, residues 264430 and 11681320 of myopalladin and residues 301385 of palladin (EMBL/GenBank/DDBJ accession number
AB023209; see Fig 4 for location of epitopes) were expressed as His6-tagged fusion proteins in the pET9D vector (
In Vitro Transcription/Translation and GST Pull-down Experiments
In vitro transcription and translation were carried out in the presence of [35S]methionine (Amersham Pharmacia Biotech) using a TNT T7-coupled reticulocyte lysate system according to the manufacturer's instructions (Promega). His6glutathione S-transferase (GST) double-tagged fusion proteins were obtained by cloning into a modified pET9D vector. The constructs were transformed into BL21-DE3 (CLONTECH Laboratories, Inc.) cells. Whole cell lysates in coating buffer (2x PBS, 1% Triton X-100) were prepared as described previously (200 µl of the lysate with 50 µl of beads (50% slurry) for 1 h at 4°C. The beads were washed three times with coating buffer and resuspended in binding buffer (20 mM Tris, pH 7.4, 100 mM KCl, 1 mM EDTA, 1% Triton X-100, plus protease inhibitors). 5 µl of in vitrotranslated 35S-labeled proteins were added to 50 µl of beads coated with 20 µg of bound GST fusion proteins in 300 µl binding buffer. The mixture was incubated for 1.5 h at 4°C, washed three times with binding buffer, and resuspended in SDS sample buffer. The protein complexes were fractionated by SDS-PAGE using 15% gels. The gels were fixed (20% methanol, 10% acetic acid), stained with Coomassie blue, treated with Amplify (Amersham Pharmacia Biotech), dried, and exposed using BioMax MR-1 film (Eastman Kodak Co.). The results of the Coomassie blue staining confirmed that equal amounts of each GST protein were bound to the beads in the different samples (data not shown).
Northern and Western Blot Analysis
For determining myopalladin and palladin's tissue-specific transcription, the full-length coding sequence of myopalladin and the subfragment bp 12894172 of palladin (EMBL/GenBank/DDBJ accession number
AB023209) was randomly labeled with 32P using the Prime-It RmT kit (Stratagene) and purified on a NucTrap column (Stratagene). The myopalladin-specific probe was hybridized to a multiple tissue Northern blot (CLONTECH Laboratories, Inc.) as described by the manufacturer. For protein expression analysis, Western blots were probed with rabbit affinity-purified antimyopalladin, antipalladin, and anti-CARP polyclonal antibodies essentially as described previously (
Cell Culture and Transfection Procedures
For myocyte expression studies, the entire myopalladin open reading frame (bp 4884450) and subfragments 4881327, 4882254, 17893299, and 33004450 (see Fig 3) were amplified by PCR and cloned into pEGFP-C1 (CLONTECH Laboratories, Inc.). Recombinant pEGFP-C1 constructs were purified using QIAGEN columns before transfection into myocytes. Plasmids were verified by sequencing. To rule out any potential artifacts resulting from the green fluorescent protein (GFP) tag, pCMVmyc-myopalladin constructs were also generated (as in
Cardiac myocytes were prepared from day 6 embryonic chick hearts and cultured as described previously (
Immunoelectron Microscopy
Cardiac muscles were dissected from the left ventricular wall of hearts rapidly excised from 89-wk-old mice (Balb/C). Muscle strips were skinned in relaxing solution containing 1% vol/vol Triton X-100 overnight at 4°C. After extensive washing with relaxing solutions, the muscles were fixed in 3% formaldehyde/PBS solution, blocked for 1 h with PBS supplemented with 1% BSA, and labeled with the primary antibodies (typically 50 µg/ml), followed by washing. Affinity-purified Fab' fragments, raised in goat against the whole rabbit IgG molecule, were used as secondary antibodies (no. 2004; Nanoprobes, Inc.). Secondary antibodies were conjugated with 1.4-nm gold particles and were further intensified with HQ silver developer for 4 min. For more details, see
Indirect Immunofluorescence Microscopy
Rat cardiac and skeletal muscle myofibrils were isolated and washed according to
Transfected cardiac myocytes were essentially stained as described (-actinin antibodies (1:1,500, EA-53; Sigma-Aldrich) or rabbit polyclonal
-actinin antibodies (1:2,000; provided by Dr. S. Craig, Johns Hopkins University, Baltimore, MD), antititin T11 monoclonal antibodies (1:1,000; Sigma-Aldrich), monoclonal antimyomesin B4 antibodies (1:20; see above), or Texas redconjugated phalloidin (Molecular Probes, Inc.) for 1 h and washed in PBS. Subsequently, the cells were stained with Texas redconjugated goat antimouse IgG+IgM (1:600) or Texas redconjugated donkey antirabbit antibodies (1:700) for 45 min. All coverslips were mounted on slides using Aqua Poly/Mount (Polysciences, Inc.) and subsequently analyzed on an Axiovert microscope (ZEISS) using 63x (NA 1.4) or 100x (NA 1.3) objectives, and micrographs were recorded as digital images on a SenSys cooled HCCD (Photometrics). Images were processed for presentation using Adobe Photoshop® 5.0. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, except the Cy2-conjugated antibodies that were purchased from Pierce Chemical Co.
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Results |
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Interaction of the COOH-terminal Region of Nebulin with Desmin and Myopalladin
To search for proteins interacting with the Z-disc region of nebulin, a fragment corresponding to its COOH-terminal 2,687 kb was amplified from rabbit psoas skeletal muscle and inserted into the yeast two-hybrid bait vector pAS2-1. The bait, referred to as pAS2-M160-M185+Ser+SH3, included the nebulin repeats, M160M176 and M182M185, the Ser-rich region, and the SH3 domain (Fig 1). The nebulin repeats M177M181 are absent from rabbit psoas muscle nebulin (
Another clone corresponded to the intermediate filament protein, desmin (
Subsequently, we performed yeast two-hybrid screens with subfragments of pAS2-M160-M185+Ser+SH3. pAS2-M160-M183 (Fig 1) identified 23 prey clones from the screening of 800,000 clones. 21 of the prey clones corresponded to desmin (
In summary, we conclude that the COOH-terminal end of nebulin, which is located 25 nm inside the Z-line lattice, specifically interacted with the novel 145-kD sarcomeric protein, myopalladin. Studies in progress are focused on characterizing further the interaction of nebulin M160M183, shown previously to be located in the periphery of the Z-line (
Analysis of Myopalladin's Primary Structure and Its Expression Patterns
Analysis of the primary structure of myopalladin revealed the presence of five Ig domains from the Ig-I subset (
The sequence homology of myopalladin to the recently described 9092-kD palladin protein from mouse is most significant, because palladin's entire sequence, including its COOH-terminal three Ig repeats and its unique NH2-terminal domain, is colinear with and conserved to myopalladin's COOH-terminal 92 kD (Fig 2 B). In particular, the COOH-terminal part of palladin containing three Ig domains is 63% identical to myopalladin's COOH-terminal region (Fig 2A and Fig B). Also highly conserved to palladin is a 15-residue box in myopalladin (residues 643658; Fig 2 B), which corresponds to myopalladin's SH3-binding site (see below). Palladin's PPPPP domains and Ser-rich domains, noted by
By searching the human genome database with myopalladin and palladin cDNAs, the myopalladin gene was shown to be derived from a single locus, located on chromosome 10q21.1 between the markers D10S207 and D10S561. Palladin was mapped to a single locus on chromosome 4q31.3 between the markers D4S1596 and D4S2910. To determine the expression pattern of the myopalladin gene, a specific probe corresponding to its full-length cDNA sequence was hybridized to a multiple tissue RNA blot. Myopalladin gene expression was found to be restricted in adult to striated muscle tissues (Fig 3). Consistent with the results of
In conclusion, myopalladin and palladin are members of the same gene family which have conserved COOH-terminal and SH3-binding domains. Palladin is encoded by a gene located on chromosome 4q31.3 and is ubiquitously expressed, whereas the myopalladin gene on chromosome 10q21.1 encodes a 145-kD protein, whose expression is restricted to striated muscle tissues (explaining the name given to this protein).
Myopalladin's Central IS3 Domain Interacts with Nebulin and Nebulette's SH3 Domain
To identify more precisely the sequences responsible for the nebulinmyopalladin interaction, nebulin and myopalladin subfragments were cotransformed into yeast (Fig 1 and Fig 4 A). Since the nebulin bait, pAS2-M160-M185+Ser+SH3, was partly autoactivating, nebulin truncations were made in the prey vector and myopalladin truncations in the bait vector. Truncations of pAS2-M160-M185+Ser+SH3 indicated that nebulin's SH3 domain alone is sufficient for the interaction with myopalladin (Fig 1). The additional presence of the Ser-rich domain, however, further enhanced yeast growth and ß-galactosidase activity and may therefore be responsible for strengthening the interaction (Fig 1).
Truncation analysis of myopalladin indicated that a 42-residue proline-rich stretch within IS3 is sufficient for the interaction with nebulin's SH3 domain (Fig 4 A). Binding sites for SH3 domains are typically proline-rich stretches of 10 amino acids, which are capable of adopting a polyproline II helix conformation (
2 (Fig 4 A), within three predicted potential SH3-binding motifs, were mutated to glycines (myopalladin-
2-mut1,2,3; Fig 4 B). Cotransformation of myopalladin-
2-mut3 with nebulin indicated that myopalladin's proline residue triplet (PPP), residues 649651, are required for binding, since this mutation abolished the interaction. The other two mutants, myopalladin-
2-mut1,2 (residues 573576 and 642645) did not affect nebulin binding (Fig 4 D). The interaction of nebulin M184+ Ser+SH3 and myopalladin-
2/myopalladin-
2-mut3 fusion peptides was also tested by GST pull-down assays. Nebulin's COOH-terminal region coprecipitated with the wild-type GSTmyopalladin-
2 but not with GST alone and only weakly with the GSTmyopalladin-
2-mut3 peptide (Fig 4 C). This confirmed that the nebulin SH3 domain specifically binds to myopalladin, involving the PPP motif, residues 649651, which is located within myopalladin's central IS3 domain (Fig 4 A).
Finally, as nebulette is thought to be a functional homologue of the Z-disc part of nebulin in the heart (2. This experiment demonstrated that the SH3 domain of nebulette also interacts with myopalladin (Fig 4 D). Therefore, both the SH3 domains of skeletal muscle nebulin and cardiac muscle nebulette specifically interact with myopalladin.
Myopalladin's COOH-terminal Region Interacts with the COOH-terminal, EF Hand Region of -Actinin
To search for other myopalladin binding partners, we screened a human skeletal muscle cDNA library with a bait containing myopalladin's full-length coding sequence (BTM-myopalladin; Fig 4 A). By screening of 1.2 x 106 colonies, 700 prey clones were identified, all of which were confirmed in the ß-galactosidase assay. 65 ß-galactosidaseexpressing clones were randomly chosen and sequenced. Three of the prey clones were derived from the COOH-terminal part of nebulin, confirming again the nebulin SH3myopalladin interaction. 13 of the prey clones contained parts of the ACTN2 gene, which encodes the
actinin-2 isoform (
actinin-2 probes to the 700 yeast colonies from the myopalladin screen, 155 clones (22%) hybridized to ACTN2. Sequence analysis of 12 ACTN2-positive clones showed that they all share a region of overlap in the COOH-terminal region of sarcomeric
actinin-2. To further narrow down the myopalladin-binding site within
actinin-2, the interaction of myopalladin with various deletion constructs of
actinin-2 (
-actinin's binding site for myopalladin is located in the COOH-terminal part of
actinin-2, from just after the spectrin repeats (before the two EF hands) up to the extreme COOH terminus. This interaction was confirmed by GST pull-down assays (Fig 5 B).
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Our two-hybrid screens isolated only actinin-2 isoforms. To test if the interaction with myopalladin was specific to the
actinin-2 isoform, we also tested
actinin-3 prey constructs, encoding a skeletal musclespecific isoform of
-actinin (
actinin-3 also interacted with myopalladin (data not shown).
To determine the sequences in myopalladin that are required for binding to -actinin, truncated myopalladin constructs were tested (Fig 4 A). Using this approach, myopalladin's
-actininbinding site was assigned to the COOH-terminal region of myopalladin. The results indicated that all three Ig domains, III+IV+V within the COOH-terminal region of myopalladin, are required for binding to
actinin-2. We conclude that myopalladin's COOH-terminal 43 kD and
-actinin's COOH-terminal EF hand region specifically interact.
Myopalladin's NH2-terminal Domain Interacts with Sarcomeric CARP
To search for proteins interacting with myopalladin's NH2-terminal domain, the NH2-terminal 1,605 kb was cloned into the yeast two-hybrid bait vector BTM117c and used to screen a human skeletal muscle cDNA library. By screening of 900,000 clones, 100 clones were identified. 88 were confirmed in the ß-galactosidase assay, and 20 were analyzed by sequencing. Six of the sequenced clones encoded the cardiac ankyrin repeat protein, CARP (
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Endogenous CARP has been detected previously in the nucleus and no sarcomeric forms have been reported (e.g.,
Localization and Assembly of Myopalladin and Palladin in Striated Muscle
To study the endogenous localization of myopalladin and palladin, specific antibodies to expressed myopalladin and palladin fragments were raised (Fig 2 A). Western blot analysis of different tissues using our affinity-purified antimyopalladin-1 antibodies detected a single band of 155 kD in rabbit cardiac, soleus, and psoas skeletal muscle (Fig 7 A). This slightly slower mobility of myopalladin observed on gels than expected from its predicted molecular mass raises the possibility of posttranslational modifications (e.g., differential phosphorylation). For palladin, a single, prominent band at
92 kD was detected with our polyclonal antipalladin antibodies in smooth muscle (
9092 and
60 kD in heart, as well as a band at
55 kD in skeletal muscle, were observed (Fig 7 B). With longer exposure times, reactivity to a faint band at
155 kD was also detected in cardiac and skeletal muscle. This could result from the cross-reactivity of our polyclonal antipalladin antibodies with the homologous myopalladin protein. However, since the palladin antibodies were raised to a region which is not homologous to myopalladin, it is not likely that the antipalladin antibodies cross-react with myopalladin. Additionally, by immunofluorescence staining the antimyopalladin and antipalladin antibodies appear not to cross-react, since both antibodies demonstrated differences in their staining patterns (Fig 7). The results from our Western blot analysis are consistent with those reported in adult tissues using monoclonal antipalladin antibodies in
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To ascertain the cellular localization of myopalladin in striated muscle, we performed immunofluorescence staining with our antimyopalladin-1specific antibodies on isolated rat heart and skeletal myofibrils. Costaining with a marker of the M-line, the myosin-associated protein myomesin, revealed that myopalladin is localized at the Z-line region in both heart and skeletal myofibrils (Fig 7 C, a' and c'); however, in some sarcomeres, a doublet of myopalladin staining is detected, indicating that this protein is also localized within the I-band (Fig 7 C, b' and d'; staining with myomesin not shown). In isolated rat cardiac myocytes, myopalladin is also detected (with varying intensities) within the nucleus of 75% cells (Fig 7 C, e'; myopalladin staining in green, myomesin staining in red). Palladin was also detected at the Z-line (but not within the I-band) in isolated rat heart and skeletal myofibrils (Fig 7 D, a' and b'; staining for myomesin not shown). No staining for palladin was detected in the nucleus in rat cardiac myocytes (Fig 7 C, c'; palladin staining in green, myomesin in red). Furthermore, palladin (Fig 7 D, c') but not myopalladin (Fig 7 C, c') was detected in I-Z-I bodies at the edges of the cardiac myocytes.
To determine the cellular localization of myopalladin when being expressed in primary cultures of chick cardiac myocytes, we transfected cells with a GFP fusion construct encoding full-length myopalladin. Full-length GFP-myopalladin was mainly targeted to Z-lines (single band in green), with some targeting to I-bands (double band in green), as demonstrated by costaining for myomesin (shown in red) (Fig 7 C, f').
Consistent with the immunofluorescence data, immunoelectron microscopy studies with myopalladin-specific antibodies detected myopalladin both within the periphery of the Z-line (Fig 8 A) and in the central I-band (Fig 8, AC). In fact, measurements of the CARP and myopalladin epitopes from the Z-disc revealed that the two proteins colocalized within the central I-band (Fig 8 D). Interestingly, for both I-bandbound myopalladin and CARP the distances of their epitopes from the Z-line were dependent on stretch (i.e., sarcomere length). In contrast, the Z-linebound myopalladin remained in a fixed position. This raises the possibility that CARP and the I-bandbound myopalladin are attached to the elastic titin filament, whereas the Z-linebound myopalladin is an integral component within Z-lines.
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Effects of Overexpression of Myopalladin on Z-Line Integrity
To investigate the functional significance of the individual domains of myopalladin in Z-line organization, we transfected chick cardiac myocytes with constructs encoding GFP fusions with full-length myopalladin, as well as four myopalladin subfragments (Fig 4 A). Two fragments corresponded to myopalladin's NH2-terminal CARP-binding region, one to its middle nebulette/nebulin-binding region, and one to its COOH-terminal -actininbinding region. We observed the effects of overexpressing these domains on the organization of
-actinin, a marker for Z-line structure, using immunofluorescence microscopy (Fig 9). Control cells expressing GFP alone exhibited a diffuse staining pattern for GFP (Fig 9 a) with mature, repeating
-actinin staining (Fig 9 b). When full-length GFP-myopalladin was overexpressed in cells, it mainly assembled in a striated pattern at the Z-line (Fig 9 c), colocalizing with
-actinin, which was assembled in a mature striated pattern (Fig 9 d), whereas some assembled at the I-band (Fig 7 C, f'). In cells overexpressing either the middle region (Fig 9 i) or the COOH-terminal region (Fig 9 k) of myopalladin, no perturbation of Z-line structure was observed (Fig 9j and Fig l, respectively). In contrast, in cells overexpressing either of two myopalladin NH2-terminal domain GFP fusion proteins (Fig 9e and Fig g), an extensively disrupted Z-line structure (i.e.,
-actinin staining) in 8085% of all transfected myocytes was observed (Fig 9f and Fig h).
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Effects of Overexpression of Myopalladin on Sarcomeric Integrity
Since the Z-lines of cells overexpressing the NH2-terminal region of myopalladin were disrupted, we next investigated the effects of overexpressing this region on overall sarcomeric integrity by observing effects on the thin, thick, and titin filament systems. Thin filament integrity was examined by staining transfected cells for actin using Texas redlabeled phalloidin, whereas thick filament organization was investigated using two markers, antimyosin and antimyomesin antibodies. Antibodies specific for a region close to the NH2-terminal (Z-line) region of titin (T11) and the COOH-terminal M-line region of titin (T114) were used to analyze the effects of the overexpression of the NH2-terminal region of myopalladin on the titin third filament system. In cells expressing GFP alone (Fig 10, a, g, and m), actin filaments (Fig 10 b), titin T11 (Fig 10 h), titin T114 (data not shown), myomesin (Fig 10 n), and myosin (data not shown) appeared in their respective typical striated pattern. Additionally, myocytes overexpressing full-length GFP-myopalladin demonstrated a striated pattern (Fig 10c, Fig i, and Fig o) and typically assembled actin (Fig 10 d), titin T11 (Fig 10 j), titin T114 (data not shown), myomesin (Fig 10 r), and myosin (data not shown). In contrast, myocytes overexpressing the GFPNH2-terminal region of myopalladin (Fig 10e, Fig k, and Fig q), exhibited a dramatic sarcomeric disruption, owing to the disrupted thin (Fig 10 f), titin (Fig 10 l), and thick (Fig 10 p) filament systems in 8085% of transfected cells. Similar results were obtained in myocytes overexpressing GFPmyopalladin-IS1, a smaller fragment of the NH2-terminal region of myopalladin that also contains the CARP-binding site (data not shown). The most likely explanation for this phenomenon is that a dominant negative phenotype occurred. That is, overexpression of the NH2-terminal region of myopalladin competed for the CARP-binding site on endogenous myopalladin. These data indicate a critical role for the NH2 terminus of myopalladin containing the CARP-binding domain for sarcomeric structure.
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Discussion |
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In this study, we aimed to further dissect the molecular components required for the coordinated organization of Z-line components into regular, hexagonal lattices. We took the approach of searching for protein interactions that anchor the COOH-terminal region of nebulin within the sarcomere. This allowed us to identify the specific binding of the nebulin modules M160M183, from the peripheral (I-band side) region of the Z-line (
This study has also led to the identification of a novel nebulin-binding protein, myopalladin, which we named based on its striking homology with the recently described ubiquitously expressed protein, palladin (-actinin in focal adhesions, stress fibers, cellcell junctions, and Z-lines, and is critical for the organization of the actin cytoskeleton and focal adhesions (
-actininbinding regions raises the intriguing possibility that the assembly of nonmuscle stress fibers and sarcomeres share common regulatory mechanisms involving myopalladin and palladin.
Previous studies have demonstrated that both the nebulette and nebulin SH3 domains target to vertebrate Z-lines when expressed in avian cardiac and skeletal myocytes, respectively (-actinin account for the targeting of the nebulin/nebulette SH3 fragments to the Z-line (
25 nm inside the Z-line. Also, myopalladin's PPP motif, and its flanking residues, are highly conserved in palladin (Fig 2 B). This strongly suggests that palladin's PPP motif can also interact with the SH3 domains of nebulin and nebulette in skeletal and cardiac muscle, respectively.
Myopalladin's COOH-terminal region, in turn, specifically binds to the EF hand motifs of -actinin. By extrapolating our data on the properties of myopalladin to what is known about palladin, we would predict that palladin's COOH-terminal three Ig domains interact with
-actinin in muscle Z-lines and in nonmuscle cells. Therefore, the data presented here, together with previous reports demonstrating the association of
-actinin with titin Z-repeats (
-actinin may form a linking system, tethering the barbed ends of the actin-thin filaments, the NH2-terminal ends of titin filaments, and the COOH-terminal ends of nebulin filaments (skeletal muscle) and nebulette (cardiac muscle) within the Z-line. Therefore, we speculate that the palladin/myopalladin
-actinin complexes are major players in assembling I-Z-I bodies in striated muscle (
-actinin interaction could provide the anchor to stabilize these structures during development, similar to palladin's proposed role in stabilizing stress fibers in nonmuscle cells (
Although myopalladin and palladin are highly homologous and are coexpressed and localized in Z-lines in mature sarcomeres (Fig 7), these two molecules appear to have very different cellular properties. Palladin, but not myopalladin, is detected in the earliest I-Z-I bodies, structures which are precursor Z-lines (for cardiac myocytes, Fig 7; for myopalladin in skeletal myogenic cells, Holtzer, H., personal communication). Possibly during myofibril assembly, some palladin complexes can be replaced by myopalladin Z-line complexes. Furthermore, myopalladin, in contrast to palladin, also localizes to the I-band and interacts with CARP (Fig 4 and Fig 7). Also, the myopalladin NH2-terminal domain is rich in potential phosphorylation sites and is efficiently phosphorylated by muscle extracts (data not shown), whereas palladin is missing these sites. Finally, palladin is extensively differentially spliced, accounting for the multiple bands detected on Western blots (this study and -actinin (data not shown). In summary, it appears that although myopalladin and palladin may interact with SH3 domaincontaining proteins via their conserved binding sites, the two proteins are likely to be regulated by different mechanisms. A molecular understanding of how the palladin/myopalladin
-actinin complex assembly is regulated is likely to improve our general understanding of Z-line assembly and maintenance.
Finally, myopalladin's NH2-terminal domain binds CARP. The CARP protein contains a nuclear localization signal, has been reported to be exclusively localized in the nucleus, and has been shown to negatively regulate the expression of cardiac genes, including the ventricular-specific myosin light chain-2, natriuretic factor, and cardiac troponin-C genes (
Here, using a novel affinity-purified CARP-specific antibody (Fig 6 B), an intranuclear localization of CARP was confirmed. However, high levels of CARP staining were also detected in the cytoplasm, as a sarcomeric bound form in the central I-band. The discovery of this large proportion of CARP that is associated with sarcomeric I-bands is likely to provide insights into understanding regulation of CARP-based signaling, since free CARP presumably is rapidly degraded by PEST-directed proteases (-actininbinding region), caused disruption of all sarcomeric proteins studied (Fig 9 and Fig 10). These data suggest that the interaction of myopalladin and CARP is critical for sarcomeric structure in cardiac myocytes. Clearly, future studies are required to investigate the molecular basis for the targeting of myopalladin and CARP to multiple cellular sites, and how these dynamic redistributions are involved in myofibril assembly, muscle structure, and muscle gene expression.
The further biochemical characterization of myopalladin, that is, its interaction with molecules crucially involved in Z-line assembly (-actinin and nebulin/nebulette) and in muscle gene expression regulation (CARP), together with an understanding of its multiple cellular localizations, may provide insights into how myofibril assembly/disassembly and gene expression regulatory mechanisms are linked.
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Footnotes |
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C. Gregorio and S. Labeit contributed equally to this work.
1 Abbreviations used in this paper: CARP, cardiac ankyrin repeat protein; GFP, green fluorescent protein; GST, glutathione S-transferase; SH, Src homology.
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Acknowledgements |
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The authors would like to thank Mark McNabb for excellent technical assistance and Dr. J. Bahl for providing us with rat cardiac myocytes.
This work was supported by grants from the Human Frontier Science Program (S. Labeit, H. Sorimachi, and C. Gregorio), CREST grant 11B-1 from the Ministry of Health and Welfare (to H. Sorimachi), National Institutes of Health grants HL57461 and HL03985 (C. Gregorio), HL07249 (A.S. McElhinny), and HL61497 and HL62881 (H. Granzier), and the Deutsche Forschungsgemeinschaft (La668/3-3 and 4-2) to S. Labeit.
Submitted: 26 December 2000
Revised: 23 February 2001
Accepted: 27 February 2001
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