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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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Interaction assays of wild-type and mutant GST-PDZ fragments and radiolabeled α-actinin 2. Autoradiogram [35S]methionine-labeled full-length α-actinin 2 after incubation and precipitation with the following GST-PDZ fragments: lane 1, GST–wild-type PDZ; lane 2, GST–G14A/W15A PDZ; lane 3, GST–H62A PDZ; lane 4, GST– L76K PDZ; lane 5, GST–L78K PDZ; lane 6, GST–L80K PDZ; lane 7, GST control. All incubations included equal amounts of input α-actinin 2. Mutation of G14A/W15A or L76K within the PDZ domain abolished binding to α-actinin 2. Exposure time for the autoradiogram was 5 h at room temperature.
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fig6: Interaction assays of wild-type and mutant GST-PDZ fragments and radiolabeled α-actinin 2. Autoradiogram [35S]methionine-labeled full-length α-actinin 2 after incubation and precipitation with the following GST-PDZ fragments: lane 1, GST–wild-type PDZ; lane 2, GST–G14A/W15A PDZ; lane 3, GST–H62A PDZ; lane 4, GST– L76K PDZ; lane 5, GST–L78K PDZ; lane 6, GST–L80K PDZ; lane 7, GST control. All incubations included equal amounts of input α-actinin 2. Mutation of G14A/W15A or L76K within the PDZ domain abolished binding to α-actinin 2. Exposure time for the autoradiogram was 5 h at room temperature.

Mentions: The phenotype of cypher mutant mice suggested that Cypher is essential for maintaining Z-line structure. One potential mechanism by which Cypher could be performing this function is to act as a linker between α-actinin and other Z-line components, thus reinforcing Z-line structure. Our previous data have shown that the non-PDZ domains of cypher do not interact with α-actinin. Do they, however, interact with other Z-line components? To investigate this possibility, we determined the localization of each non-PDZ domain in cultured neonatal rat cardiomyocytes. A cDNA encoding green fluorescent protein (GFP) was fused to full-length Cypher1, Cypher2, and each distinct domain within these Cypher proteins. A GFP fusion construct was also made for a mutant PDZ domain which no longer binds to α-actinin 2 (L78K; see Fig. 6). Resulting constructs were then transfected into rat neonatal cardiomyocytes. 2 d after transfection, cells were immunostained with anti–α-actinin 2 antibody. Antibody staining and GFP expression were visualized by fluorescence microscopy (Fig. 5).


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)

Interaction assays of wild-type and mutant GST-PDZ fragments and radiolabeled α-actinin 2. Autoradiogram [35S]methionine-labeled full-length α-actinin 2 after incubation and precipitation with the following GST-PDZ fragments: lane 1, GST–wild-type PDZ; lane 2, GST–G14A/W15A PDZ; lane 3, GST–H62A PDZ; lane 4, GST– L76K PDZ; lane 5, GST–L78K PDZ; lane 6, GST–L80K PDZ; lane 7, GST control. All incubations included equal amounts of input α-actinin 2. Mutation of G14A/W15A or L76K within the PDZ domain abolished binding to α-actinin 2. Exposure time for the autoradiogram was 5 h at room temperature.
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Related In: Results  -  Collection

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fig6: Interaction assays of wild-type and mutant GST-PDZ fragments and radiolabeled α-actinin 2. Autoradiogram [35S]methionine-labeled full-length α-actinin 2 after incubation and precipitation with the following GST-PDZ fragments: lane 1, GST–wild-type PDZ; lane 2, GST–G14A/W15A PDZ; lane 3, GST–H62A PDZ; lane 4, GST– L76K PDZ; lane 5, GST–L78K PDZ; lane 6, GST–L80K PDZ; lane 7, GST control. All incubations included equal amounts of input α-actinin 2. Mutation of G14A/W15A or L76K within the PDZ domain abolished binding to α-actinin 2. Exposure time for the autoradiogram was 5 h at room temperature.
Mentions: The phenotype of cypher mutant mice suggested that Cypher is essential for maintaining Z-line structure. One potential mechanism by which Cypher could be performing this function is to act as a linker between α-actinin and other Z-line components, thus reinforcing Z-line structure. Our previous data have shown that the non-PDZ domains of cypher do not interact with α-actinin. Do they, however, interact with other Z-line components? To investigate this possibility, we determined the localization of each non-PDZ domain in cultured neonatal rat cardiomyocytes. A cDNA encoding green fluorescent protein (GFP) was fused to full-length Cypher1, Cypher2, and each distinct domain within these Cypher proteins. A GFP fusion construct was also made for a mutant PDZ domain which no longer binds to α-actinin 2 (L78K; see Fig. 6). Resulting constructs were then transfected into rat neonatal cardiomyocytes. 2 d after transfection, cells were immunostained with anti–α-actinin 2 antibody. Antibody staining and GFP expression were visualized by fluorescence microscopy (Fig. 5).

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