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Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

Bang ML, Mudry RE, McElhinny AS, Trombitás K, Geach AJ, Yamasaki R, Sorimachi H, Granzier H, Gregorio CC, Labeit S - J. Cell Biol. (2001)

Bottom Line: 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.Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP 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 alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg 69117, Germany.

ABSTRACT
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 alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding 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. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-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 NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP 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 alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

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Overexpression of the NH2-terminal region of myopalladin in cardiac myocytes results in disruption of thin, titin, and thick filaments. Chick cardiac myocytes expressing GFP alone (GFP; a, g, and m), GFP–full-length myopalladin (GFP-myopalladin; c, i, and o), and GFP–NH2-terminal myopalladin region (GFP–myopalladin-N; e, k and p) were fixed 3–5 d after transfection and stained with Texas red–conjugated phalloidin (b, d, and f), antititin T11 antibodies followed by Texas red–conjugated secondary antibodies (h, j, and l), or antimyomesin antibodies followed by Texas red–conjugated secondary antibodies (n, p, and r). Arrows point to typical striated staining pattern of actin (b and d), titin (h and j), and myomesin (n and p); arrowheads point to disrupted actin (f), titin (l), and myomesin (r). Bar, 10 μm.
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Figure 10: Overexpression of the NH2-terminal region of myopalladin in cardiac myocytes results in disruption of thin, titin, and thick filaments. Chick cardiac myocytes expressing GFP alone (GFP; a, g, and m), GFP–full-length myopalladin (GFP-myopalladin; c, i, and o), and GFP–NH2-terminal myopalladin region (GFP–myopalladin-N; e, k and p) were fixed 3–5 d after transfection and stained with Texas red–conjugated phalloidin (b, d, and f), antititin T11 antibodies followed by Texas red–conjugated secondary antibodies (h, j, and l), or antimyomesin antibodies followed by Texas red–conjugated secondary antibodies (n, p, and r). Arrows point to typical striated staining pattern of actin (b and d), titin (h and j), and myomesin (n and p); arrowheads point to disrupted actin (f), titin (l), and myomesin (r). Bar, 10 μm.

Mentions: 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 red–labeled 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 GFP–NH2-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 80–85% of transfected cells. Similar results were obtained in myocytes overexpressing GFP–myopalladin-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.


Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

Bang ML, Mudry RE, McElhinny AS, Trombitás K, Geach AJ, Yamasaki R, Sorimachi H, Granzier H, Gregorio CC, Labeit S - J. Cell Biol. (2001)

Overexpression of the NH2-terminal region of myopalladin in cardiac myocytes results in disruption of thin, titin, and thick filaments. Chick cardiac myocytes expressing GFP alone (GFP; a, g, and m), GFP–full-length myopalladin (GFP-myopalladin; c, i, and o), and GFP–NH2-terminal myopalladin region (GFP–myopalladin-N; e, k and p) were fixed 3–5 d after transfection and stained with Texas red–conjugated phalloidin (b, d, and f), antititin T11 antibodies followed by Texas red–conjugated secondary antibodies (h, j, and l), or antimyomesin antibodies followed by Texas red–conjugated secondary antibodies (n, p, and r). Arrows point to typical striated staining pattern of actin (b and d), titin (h and j), and myomesin (n and p); arrowheads point to disrupted actin (f), titin (l), and myomesin (r). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 10: Overexpression of the NH2-terminal region of myopalladin in cardiac myocytes results in disruption of thin, titin, and thick filaments. Chick cardiac myocytes expressing GFP alone (GFP; a, g, and m), GFP–full-length myopalladin (GFP-myopalladin; c, i, and o), and GFP–NH2-terminal myopalladin region (GFP–myopalladin-N; e, k and p) were fixed 3–5 d after transfection and stained with Texas red–conjugated phalloidin (b, d, and f), antititin T11 antibodies followed by Texas red–conjugated secondary antibodies (h, j, and l), or antimyomesin antibodies followed by Texas red–conjugated secondary antibodies (n, p, and r). Arrows point to typical striated staining pattern of actin (b and d), titin (h and j), and myomesin (n and p); arrowheads point to disrupted actin (f), titin (l), and myomesin (r). Bar, 10 μm.
Mentions: 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 red–labeled 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 GFP–NH2-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 80–85% of transfected cells. Similar results were obtained in myocytes overexpressing GFP–myopalladin-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.

Bottom Line: 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.Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP 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 alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg 69117, Germany.

ABSTRACT
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 alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding 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. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-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 NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP 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 alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Show MeSH