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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).

Show MeSH
Northern blot analysis reveals that detectable myopalladin gene expression is restricted to striated muscle. Myopalladin- and palladin-specific cDNA probes were hybridized to human RNA master blots (CLONTECH Laboratories, Inc.). Left, myopalladin gene expression is restricted to skeletal muscle (C3), fetal heart (G2), and adult heart (C1). Right, palladin transcripts are detected in all human tissues tested and are particularly abundant in striated and smooth muscle tissues (e.g., bladder, uterus, prostate, and stomach, C5–C8, respectively).
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Figure 3: Northern blot analysis reveals that detectable myopalladin gene expression is restricted to striated muscle. Myopalladin- and palladin-specific cDNA probes were hybridized to human RNA master blots (CLONTECH Laboratories, Inc.). Left, myopalladin gene expression is restricted to skeletal muscle (C3), fetal heart (G2), and adult heart (C1). Right, palladin transcripts are detected in all human tissues tested and are particularly abundant in striated and smooth muscle tissues (e.g., bladder, uterus, prostate, and stomach, C5–C8, respectively).

Mentions: For myocyte expression studies, the entire myopalladin open reading frame (bp 488–4450) and subfragments 488–1327, 488–2254, 1789–3299, and 3300–4450 (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 Gregorio et al. 1998) and tested in overexpression studies: identical results were obtained (data not shown).


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)

Northern blot analysis reveals that detectable myopalladin gene expression is restricted to striated muscle. Myopalladin- and palladin-specific cDNA probes were hybridized to human RNA master blots (CLONTECH Laboratories, Inc.). Left, myopalladin gene expression is restricted to skeletal muscle (C3), fetal heart (G2), and adult heart (C1). Right, palladin transcripts are detected in all human tissues tested and are particularly abundant in striated and smooth muscle tissues (e.g., bladder, uterus, prostate, and stomach, C5–C8, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169455&req=5

Figure 3: Northern blot analysis reveals that detectable myopalladin gene expression is restricted to striated muscle. Myopalladin- and palladin-specific cDNA probes were hybridized to human RNA master blots (CLONTECH Laboratories, Inc.). Left, myopalladin gene expression is restricted to skeletal muscle (C3), fetal heart (G2), and adult heart (C1). Right, palladin transcripts are detected in all human tissues tested and are particularly abundant in striated and smooth muscle tissues (e.g., bladder, uterus, prostate, and stomach, C5–C8, respectively).
Mentions: For myocyte expression studies, the entire myopalladin open reading frame (bp 488–4450) and subfragments 488–1327, 488–2254, 1789–3299, and 3300–4450 (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 Gregorio et al. 1998) and tested in overexpression studies: identical results were obtained (data not shown).

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