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

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.

Figure 1.

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).

Figure 2.

X-gal staining of cypher LacZ/knock-in heterozygous embryos at different development stages. Embryos from embryonic day (E) 8.5, 9.5, 11.5, and 13.5 were stained with X-gal for ∼8 h at 30°C. X-gal staining of heart (H) was detected from E 8.5. Somite (S) staining was not detectable at E 8.5, but was detected at E 9.5 in a decreasing rostral to caudal gradient. 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 (M). At E 13.5, cypher expression was detected throughout the embryonic musculature. Bar, 1 mm.

Figure 3.

Histological analyses of skeletal and cardiac muscles from cypher ^−/− and their wild-type littermate controls. Sagittal sections of postnatal day 1 wild-type (+/+) and cypher knockout mice (−/−) were stained with hematoxylin and eosin. Note the ventricular dilation in the knockout mice. RV, right ventricle; LV, left ventricle. Bar, 1 mm.

Figure 4.

Ultrastructural analysis of diaphragm muscle and heart architecture as assessed by TEM. Representative images from diaphragm muscle of E 17.5 and day 1 neonatal (N1) wild-type (+/+) and cypher knockout (−/−) mice (A–D). A comparison of diaphragm muscle from wild-type and knockout mice at E 17.5, when diaphragm muscle is inactive, shows well-preserved Z-line and M line structure (A and B). In contrast, Z-lines in diaphragm muscle from knockout mice at postnatal day 1 are severely disorganized and disrupted; M-lines, however, are relatively normal (C and D). Representative images from cardiac muscle of E 17.5 and day 1 neonatal wild-type and cypher knockout mice (E–H). In embryonic cardiac muscle, Z lines are evident, but distinct, well-formed M lines are not yet present (Anversa et al., 1981). In Cypher knockout mice, E 17.5 cardiac muscle, which has been functional since E 8, exhibits fragmented and disorganized Z-lines (E and F). In postnatal day 1 cardiac muscle of wild-type mice, both Z-lines and M-lines are well formed and distinctly visible. In contrast, only remnants of the Z-line and no M-lines are visible in cardiac muscle from mutant mice (G and H). Bar, 1.5 μm.

Figure 6.

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.

Figure 5.

Fluorescence microscopy of neonatal rat cardiomyocytes transfected with GFP fusions of distinct Cypher domains. Transfected rat neonatal cardiomyocytes were stained after 2 d in culture: lane 1, GFP fusion proteins (green); lane 2, rhodamine staining for α-actinin 2 (red); lane 3, superimposition of the two images (orange). (A) GFP alone; (B) Cypher1; (C) Cypher2; (D) PDZ domain (aa 1–84 of Cypher); (E) YS/TPS/TP repeat-containing region (aa 85–546 of Cypher1); (F). LIM domains (aa 547–723 of Cypher1); (G) PDZ-less Cypher2 (aa 85–288 of Cypher2); and (H) L78K mutant PDZ domain. Each distinct domain of Cypher1 and Cypher2, including the PDZ domain, independently localized to the Z-line (B–G). Disruption of α-actinin 2 binding by mutation of L78K within the Cypher PDZ domain prevented exclusive localization to the Z-line (H). Bar, 10 μm.

Figure 5.

Fluorescence microscopy of neonatal rat cardiomyocytes transfected with GFP fusions of distinct Cypher domains. Transfected rat neonatal cardiomyocytes were stained after 2 d in culture: lane 1, GFP fusion proteins (green); lane 2, rhodamine staining for α-actinin 2 (red); lane 3, superimposition of the two images (orange). (A) GFP alone; (B) Cypher1; (C) Cypher2; (D) PDZ domain (aa 1–84 of Cypher); (E) YS/TPS/TP repeat-containing region (aa 85–546 of Cypher1); (F). LIM domains (aa 547–723 of Cypher1); (G) PDZ-less Cypher2 (aa 85–288 of Cypher2); and (H) L78K mutant PDZ domain. Each distinct domain of Cypher1 and Cypher2, including the PDZ domain, independently localized to the Z-line (B–G). Disruption of α-actinin 2 binding by mutation of L78K within the Cypher PDZ domain prevented exclusive localization to the Z-line (H). Bar, 10 μm.

Figure 7.

Interaction of the PDZ domain of Cypher requires the COOH terminus of α-actinin. (A) Schematic diagram of α-actinin 2. The actin binding domain (ABD, aa 1–273), spectrin-like repeats (SLR, aa 274–755), and EF hands (aa 755–894) are indicated. B. Autoradiogram of ³⁵S methionine-labeled proteins after coprecipitated with GST-PDZ glutathione resin (see Materials and Methods). Lane 1, full-length α-actinin 2; lane 2, Spectrin repeats; lane 3, ABD domain; lane 4, the four EF hands. Exposure time for the autoradiogram was 4 h at room temperature. The bottom panel shows the input of each in vitro translated protein. Exposure time for the autoradiogram was 20 min at room temperature. Strongest binding to the PDZ domain was observed with the COOH-terminal four EF hands of α-actinin 2 (lane 4). C. Autoradiogram of ³⁵S methionine-labeled α-actinin 2 COOH-terminal fragments after coincubation and precipitation with GST-PDZ fusion proteins. Lane 1, ³⁵S- labeled four EF hands (4EF, aa 756 to 894); lane 2, four EF hands deleted for the three COOH-terminal amino acids (4EF-del3, aa 756 to 891); lane 3, first two EF hands (EF1-2, aa 756–821); lane 4, last two EF hands (EF3-4, aa 822–893). Exposure time for the autoradiogram was 3 h at room temperature. The bottom panel shows 20% input of in vitro–translated protein. Exposure time for the autoradiogram was 30 min at room temperature. Deletion of the last two EF hands, including the three terminal amino acids, or deletion of the three terminal amino acids alone, abolished binding to the PDZ domain (lanes 2 and 3). (D) Autoradiogram of [³⁵S]methionine-labeled proteins after coprecipitation with GST-PDZ glutathione resin. Lane 1, full-length α-actinin 2; lane 2, α-actinin 2 deleted for the three COOH-terminal amino acids (actinin-del3); lane 3 and 4, 20% input of in vitro–translated full-length α-actinin 2 and actinin-del3 proteins. Exposure time for the autoradiogram was 3 h at room temperature.