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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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Characterization of endogenous myopalladins and palladins in striated muscle. (A) Specificity of affinity-purified antimyopalladin antibodies. Antimyopalladin antibodies recognize a band at ∼155 kD in rabbit heart (lane 1), soleus (lane 2), and psoas muscle (lane 3) by Western blot analysis. Note, on some blots an ∼35-kD band was also detected; the detection of this band was variable and its significance is unknown. (B) Specificity of affinity-purified antipalladin antibodies. Antipalladin antibodies recognize a 92-kD band in rat smooth muscle from the small intestine (lane 3), as well as multiple bands in heart (lane 1) and skeletal muscle (lane 2). (C) Immunofluorescence staining of myopalladin in washed, isolated myofibrils from rat heart (a′ and b′) and skeletal (c′ and d′) muscle, as well as in primary cultures of rat cardiac myocytes (e′; myopalladin staining in green, myomesin staining in red) demonstrating that myopalladin can be detected as a single striation at the Z-line (a′ and c′, arrows) and as a doublet within the I-band (in close proximity to the Z-line) (b′ and d′, double arrows). Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antimyopalladin-1 antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′–d′) followed by Texas red–conjugated secondary antibodies. Note the additional staining of myopalladin in the nucleus in e′. Arrowheads in e′ mark the absence of detectable myopalladin (and myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. (f′) Immunofluorescence image demonstrating the targeting of expressed GFP-myopalladin to the Z-line and to the I-band in primary cultures of chick cardiac myocytes. Cardiomyocytes expressing GFP–full-length myopalladin were fixed 3–5 d after transfection and stained with antimyomesin antibodies followed by Texas red–conjugated secondary antibodies and analyzed by immunofluorescence microscopy. Single arrows mark Z-line staining, whereas double arrows mark I-band staining. Significant variability in the relative labeling intensities of myopalladin at the Z-line vs. the I-band was observed. N, nucleus. (D) Immunofluorescence staining of palladin in washed, isolated myofibrils from rat heart (a′) and skeletal (b′) muscle, as well as in primary cultures of rat cardiac myocytes (c′; palladin staining in green, myomesin staining in red), demonstrating that palladin is localized at the Z-line. Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antipalladin antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′ and b′) followed by Texas red–conjugated secondary antibodies. Arrowheads in c′ mark the presence of palladin (but not myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. Note that palladin staining was not detected in the nucleus in c′. N, nucleus. Bars, 10 μm.
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Figure 7: Characterization of endogenous myopalladins and palladins in striated muscle. (A) Specificity of affinity-purified antimyopalladin antibodies. Antimyopalladin antibodies recognize a band at ∼155 kD in rabbit heart (lane 1), soleus (lane 2), and psoas muscle (lane 3) by Western blot analysis. Note, on some blots an ∼35-kD band was also detected; the detection of this band was variable and its significance is unknown. (B) Specificity of affinity-purified antipalladin antibodies. Antipalladin antibodies recognize a 92-kD band in rat smooth muscle from the small intestine (lane 3), as well as multiple bands in heart (lane 1) and skeletal muscle (lane 2). (C) Immunofluorescence staining of myopalladin in washed, isolated myofibrils from rat heart (a′ and b′) and skeletal (c′ and d′) muscle, as well as in primary cultures of rat cardiac myocytes (e′; myopalladin staining in green, myomesin staining in red) demonstrating that myopalladin can be detected as a single striation at the Z-line (a′ and c′, arrows) and as a doublet within the I-band (in close proximity to the Z-line) (b′ and d′, double arrows). Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antimyopalladin-1 antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′–d′) followed by Texas red–conjugated secondary antibodies. Note the additional staining of myopalladin in the nucleus in e′. Arrowheads in e′ mark the absence of detectable myopalladin (and myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. (f′) Immunofluorescence image demonstrating the targeting of expressed GFP-myopalladin to the Z-line and to the I-band in primary cultures of chick cardiac myocytes. Cardiomyocytes expressing GFP–full-length myopalladin were fixed 3–5 d after transfection and stained with antimyomesin antibodies followed by Texas red–conjugated secondary antibodies and analyzed by immunofluorescence microscopy. Single arrows mark Z-line staining, whereas double arrows mark I-band staining. Significant variability in the relative labeling intensities of myopalladin at the Z-line vs. the I-band was observed. N, nucleus. (D) Immunofluorescence staining of palladin in washed, isolated myofibrils from rat heart (a′) and skeletal (b′) muscle, as well as in primary cultures of rat cardiac myocytes (c′; palladin staining in green, myomesin staining in red), demonstrating that palladin is localized at the Z-line. Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antipalladin antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′ and b′) followed by Texas red–conjugated secondary antibodies. Arrowheads in c′ mark the presence of palladin (but not myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. Note that palladin staining was not detected in the nucleus in c′. N, nucleus. Bars, 10 μm.

Mentions: 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 (Parast and Otey 2000), whereas bands at ∼90–92 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 Parast and Otey 2000, with the exception that our polyclonal antipalladin antibodies appeared to recognize more palladin-related proteins (although the smaller proteins we detected may not have been resolved on their gel system).


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)

Characterization of endogenous myopalladins and palladins in striated muscle. (A) Specificity of affinity-purified antimyopalladin antibodies. Antimyopalladin antibodies recognize a band at ∼155 kD in rabbit heart (lane 1), soleus (lane 2), and psoas muscle (lane 3) by Western blot analysis. Note, on some blots an ∼35-kD band was also detected; the detection of this band was variable and its significance is unknown. (B) Specificity of affinity-purified antipalladin antibodies. Antipalladin antibodies recognize a 92-kD band in rat smooth muscle from the small intestine (lane 3), as well as multiple bands in heart (lane 1) and skeletal muscle (lane 2). (C) Immunofluorescence staining of myopalladin in washed, isolated myofibrils from rat heart (a′ and b′) and skeletal (c′ and d′) muscle, as well as in primary cultures of rat cardiac myocytes (e′; myopalladin staining in green, myomesin staining in red) demonstrating that myopalladin can be detected as a single striation at the Z-line (a′ and c′, arrows) and as a doublet within the I-band (in close proximity to the Z-line) (b′ and d′, double arrows). Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antimyopalladin-1 antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′–d′) followed by Texas red–conjugated secondary antibodies. Note the additional staining of myopalladin in the nucleus in e′. Arrowheads in e′ mark the absence of detectable myopalladin (and myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. (f′) Immunofluorescence image demonstrating the targeting of expressed GFP-myopalladin to the Z-line and to the I-band in primary cultures of chick cardiac myocytes. Cardiomyocytes expressing GFP–full-length myopalladin were fixed 3–5 d after transfection and stained with antimyomesin antibodies followed by Texas red–conjugated secondary antibodies and analyzed by immunofluorescence microscopy. Single arrows mark Z-line staining, whereas double arrows mark I-band staining. Significant variability in the relative labeling intensities of myopalladin at the Z-line vs. the I-band was observed. N, nucleus. (D) Immunofluorescence staining of palladin in washed, isolated myofibrils from rat heart (a′) and skeletal (b′) muscle, as well as in primary cultures of rat cardiac myocytes (c′; palladin staining in green, myomesin staining in red), demonstrating that palladin is localized at the Z-line. Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antipalladin antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′ and b′) followed by Texas red–conjugated secondary antibodies. Arrowheads in c′ mark the presence of palladin (but not myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. Note that palladin staining was not detected in the nucleus in c′. N, nucleus. Bars, 10 μm.
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Figure 7: Characterization of endogenous myopalladins and palladins in striated muscle. (A) Specificity of affinity-purified antimyopalladin antibodies. Antimyopalladin antibodies recognize a band at ∼155 kD in rabbit heart (lane 1), soleus (lane 2), and psoas muscle (lane 3) by Western blot analysis. Note, on some blots an ∼35-kD band was also detected; the detection of this band was variable and its significance is unknown. (B) Specificity of affinity-purified antipalladin antibodies. Antipalladin antibodies recognize a 92-kD band in rat smooth muscle from the small intestine (lane 3), as well as multiple bands in heart (lane 1) and skeletal muscle (lane 2). (C) Immunofluorescence staining of myopalladin in washed, isolated myofibrils from rat heart (a′ and b′) and skeletal (c′ and d′) muscle, as well as in primary cultures of rat cardiac myocytes (e′; myopalladin staining in green, myomesin staining in red) demonstrating that myopalladin can be detected as a single striation at the Z-line (a′ and c′, arrows) and as a doublet within the I-band (in close proximity to the Z-line) (b′ and d′, double arrows). Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antimyopalladin-1 antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′–d′) followed by Texas red–conjugated secondary antibodies. Note the additional staining of myopalladin in the nucleus in e′. Arrowheads in e′ mark the absence of detectable myopalladin (and myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. (f′) Immunofluorescence image demonstrating the targeting of expressed GFP-myopalladin to the Z-line and to the I-band in primary cultures of chick cardiac myocytes. Cardiomyocytes expressing GFP–full-length myopalladin were fixed 3–5 d after transfection and stained with antimyomesin antibodies followed by Texas red–conjugated secondary antibodies and analyzed by immunofluorescence microscopy. Single arrows mark Z-line staining, whereas double arrows mark I-band staining. Significant variability in the relative labeling intensities of myopalladin at the Z-line vs. the I-band was observed. N, nucleus. (D) Immunofluorescence staining of palladin in washed, isolated myofibrils from rat heart (a′) and skeletal (b′) muscle, as well as in primary cultures of rat cardiac myocytes (c′; palladin staining in green, myomesin staining in red), demonstrating that palladin is localized at the Z-line. Isolated myofibrils and cardiac myocytes were labeled with affinity-purified antipalladin antibodies, followed by Cy2-conjugated secondary antibodies, and with antimyomesin antibodies (data not shown in a′ and b′) followed by Texas red–conjugated secondary antibodies. Arrowheads in c′ mark the presence of palladin (but not myomesin) staining in I-Z-I bodies, located at the edges of cultured cardiac myocytes. Note that palladin staining was not detected in the nucleus in c′. N, nucleus. Bars, 10 μm.
Mentions: 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 (Parast and Otey 2000), whereas bands at ∼90–92 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 Parast and Otey 2000, with the exception that our polyclonal antipalladin antibodies appeared to recognize more palladin-related proteins (although the smaller proteins we detected may not have been resolved on their gel system).

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
Related in: MedlinePlus