Limits...
Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.

Zhou Q, Chu PH, Huang C, Cheng CF, Martone ME, Knoll G, Shelton GD, Evans S, Chen J - J. Cell Biol. (2001)

Bottom Line: Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function.In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components.These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Medicine, University of California at San Diego School of Medicine, La Jolla, CA 92093, USA.

ABSTRACT
Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH(2) terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

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Targeting the cypher gene. (A) Targeting strategy. A restriction map of the relevant genomic region of cypher is shown on top, the targeting construct is shown in the center, and the mutated locus after recombination is shown at the bottom. ATG is the translational start site. The arrow indicates the orientation of β-galactosidase cDNA and neomycin resistance gene. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI; X, XbaI. β-Galactosidase cDNA; and neo, neomycin resistance gene. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNAs from electroporated ES cells were digested with BamHI and analyzed by Southern blot analysis with probe as shown in A. The 5- and 3-kb bands represent wild-type and targeted allele, respectively. (C) Detection of Cypher protein by Western blot analysis. Proteins prepared from neonatal day 1 skeletal muscle of wild-type (+/+) and cypher knockout mice (−/−) were analyzed with anti-Cypher (top) and antitropomyosin C monoclonal antibodies (bottom).
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fig1: Targeting the cypher gene. (A) Targeting strategy. A restriction map of the relevant genomic region of cypher is shown on top, the targeting construct is shown in the center, and the mutated locus after recombination is shown at the bottom. ATG is the translational start site. The arrow indicates the orientation of β-galactosidase cDNA and neomycin resistance gene. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI; X, XbaI. β-Galactosidase cDNA; and neo, neomycin resistance gene. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNAs from electroporated ES cells were digested with BamHI and analyzed by Southern blot analysis with probe as shown in A. The 5- and 3-kb bands represent wild-type and targeted allele, respectively. (C) Detection of Cypher protein by Western blot analysis. Proteins prepared from neonatal day 1 skeletal muscle of wild-type (+/+) and cypher knockout mice (−/−) were analyzed with anti-Cypher (top) and antitropomyosin C monoclonal antibodies (bottom).

Mentions: To understand the in vivo function of Cypher in striated muscle and to exploit cypher as a potentially useful lineage marker, we used homologous recombination to knock-in a β-galactosidase cDNA downstream of the endogenous cypher promoter, meanwhile disrupting the cypher gene which encodes both Cypher1 and Cypher2 (Fig. 1). Assays for β-galactosidase activity are very sensitive and give resolution at the single cell level, thus permitting us to look in greater detail at expression of cypher during embryogenesis (Chu et al., 2000). As shown in Fig. 2, cypher is expressed exclusively in heart at embryonic day (E) 8.5, but at E 9.5 it was detected in somites in a rostral to caudal gradient, with highest expression rostrally. The somite expression pattern progresses caudally through E 11.5, corresponding to somite maturation. At this stage, cypher expression was also detected in cells migrating from the myotome. At E 13.5, cypher expression was detected throughout the embryonic musculature. The X-gal staining pattern observed here matches well with our previous RNA in situ hybridization results, with the exception that the X-gal staining was observed earlier in the somites, in keeping with its greater sensitivity (Zhou et al., 1999).


Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.

Zhou Q, Chu PH, Huang C, Cheng CF, Martone ME, Knoll G, Shelton GD, Evans S, Chen J - J. Cell Biol. (2001)

Targeting the cypher gene. (A) Targeting strategy. A restriction map of the relevant genomic region of cypher is shown on top, the targeting construct is shown in the center, and the mutated locus after recombination is shown at the bottom. ATG is the translational start site. The arrow indicates the orientation of β-galactosidase cDNA and neomycin resistance gene. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI; X, XbaI. β-Galactosidase cDNA; and neo, neomycin resistance gene. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNAs from electroporated ES cells were digested with BamHI and analyzed by Southern blot analysis with probe as shown in A. The 5- and 3-kb bands represent wild-type and targeted allele, respectively. (C) Detection of Cypher protein by Western blot analysis. Proteins prepared from neonatal day 1 skeletal muscle of wild-type (+/+) and cypher knockout mice (−/−) were analyzed with anti-Cypher (top) and antitropomyosin C monoclonal antibodies (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198871&req=5

fig1: Targeting the cypher gene. (A) Targeting strategy. A restriction map of the relevant genomic region of cypher is shown on top, the targeting construct is shown in the center, and the mutated locus after recombination is shown at the bottom. ATG is the translational start site. The arrow indicates the orientation of β-galactosidase cDNA and neomycin resistance gene. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI; X, XbaI. β-Galactosidase cDNA; and neo, neomycin resistance gene. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNAs from electroporated ES cells were digested with BamHI and analyzed by Southern blot analysis with probe as shown in A. The 5- and 3-kb bands represent wild-type and targeted allele, respectively. (C) Detection of Cypher protein by Western blot analysis. Proteins prepared from neonatal day 1 skeletal muscle of wild-type (+/+) and cypher knockout mice (−/−) were analyzed with anti-Cypher (top) and antitropomyosin C monoclonal antibodies (bottom).
Mentions: To understand the in vivo function of Cypher in striated muscle and to exploit cypher as a potentially useful lineage marker, we used homologous recombination to knock-in a β-galactosidase cDNA downstream of the endogenous cypher promoter, meanwhile disrupting the cypher gene which encodes both Cypher1 and Cypher2 (Fig. 1). Assays for β-galactosidase activity are very sensitive and give resolution at the single cell level, thus permitting us to look in greater detail at expression of cypher during embryogenesis (Chu et al., 2000). As shown in Fig. 2, cypher is expressed exclusively in heart at embryonic day (E) 8.5, but at E 9.5 it was detected in somites in a rostral to caudal gradient, with highest expression rostrally. The somite expression pattern progresses caudally through E 11.5, corresponding to somite maturation. At this stage, cypher expression was also detected in cells migrating from the myotome. At E 13.5, cypher expression was detected throughout the embryonic musculature. The X-gal staining pattern observed here matches well with our previous RNA in situ hybridization results, with the exception that the X-gal staining was observed earlier in the somites, in keeping with its greater sensitivity (Zhou et al., 1999).

Bottom Line: Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function.In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components.These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Medicine, University of California at San Diego School of Medicine, La Jolla, CA 92093, USA.

ABSTRACT
Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH(2) terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

Show MeSH
Related in: MedlinePlus