Figure 1.
MHC is critical for muscle striation formation.
(A) Schematic organization of a myofibril, represented here with two sarcomeres. Sarcomeres are defined as the segment between two neighboring Z-lines. Thin filaments include actin filaments and their associated proteins such as troponins (Tns) (TnC, TnI, and TnT) and tropomyosin (Tm). Actin filaments are the major components of I-bands, and are cross-linked to Z-lines via α-actinin. Thick filaments are composed of myosin and are connected from the M-line to the Z-line by titin. A number of proteins important for the stability of sarcomeres, such as zipper and Zasp, are found in the Z-line. (B) Confocal fluorescent micrographs of control muscles of a stage 17 wild-type embryo (top panels) and myosin heavy chain (Mhc) amorphic mutant muscles from Mhc1 of same stage (bottom panels) stained by phalloidin (blue in merge), anti-α-actinin (red in merge) and anti-β-integrin (green in merge). Note that there is no obvious striation in Mhc null mutant muscles, and that β-integrin staining does not align with that of α-actinin. (C) Confocal images of control muscles of a stage 17 wild-type embryo (top panels) and Mhc1 of same stage (bottom panels) stained by anti-muscle MHC (blue in merge), anti-α-actinin (red in merge) and anti-zipper (green in merge). Note that in wild-type muscles, zipper colocalizes with α-actinin as shown in yellow in the merged image, but not with MHC. In addition, rat-anti-MHC was able to detect truncated MHC fragments in Mhc1 mutant muscles, and its staining overlaps with actin. This staining most likely reflects the ability of the Subfragment 1 region of MHC to bind to actin filaments. Scale bars: 10 µm.
Figure 2.
Tns-Tm complexes play an important role in sarcomere assembly.
(A,B) Primary muscle cells were isolated from Oregon R embryos and treated with dsRNA against lacZ (A and B, upper panels), TnT (A. lower panels), or TnC (B. lower panels). Sarcomeric organization was evaluated following staining using polyclonal antibodies against MHC, α-actinin or actin. The efficacy of TnT and TnC RNAi knock-downs were evaluated using anti-TnT and anti-TnC specific antibodies, respectively. Note that while removal of TnT has severe effects on the striated organization of the sarcomere, depletion of TnC has little effect. (C) To determine the relationships between TnT, TnI, TnC and Tm, primary muscle cells were treated with TnT, TnI, TnC or Tm RNAi and stained with antibodies against TnT, TnI, TnC and Tm, respectively. Note the severe effects on the striated pattern in the absence of TnT, TnI and Tm, but not TnC. Scale bars: 10 µm.
Figure 3.
The I-Z-I complex is required for sarcomere organization.
(A–D) DsRNAs against actin (A), α–actinin (B), zipper (C), Zasp (D) or control lacZ were applied to primary muscle cell cultures. Phalloidin, anti-α-actinin, anti-zipper and anti-Zasp antibodies revealed the knock-down efficiencies of the various dsRNAs. The structure of the muscles was monitored following staining using an anti-MHC antibody. Note that in the absence of a single component of I-Z-I, thick filament remains in a striated pattern. As a control, we treated muscle cells with actin RNAi in combination with lacZ RNAi or mlp84B RNAi, which did not produce any significant effects on muscle striation pattern (data not shown). (E) DsRNAs against α-actinin, zipper and Zasp were individually mixed with a dsRNA against actin in a 1∶1 ratio. The sarcomeric organization of primary muscle cells was analyzed following staining using anti-MHC, anti-α-actinin and anti-actin antibodies, respectively. Note the absence of MHC striation when two I-Z-I components are depleted. We also confirmed the localization pattern of Z-line proteins with our available antibodies after double knock-down and showed absence of striation (data not shown), indicating that the I-Z-I complex is critical for the periodic localization of other functional complexes such as MHC and Tns-Tm. Scale bars: 10 µm.
Figure 4.
Zipper/Zasp/α-actinin acts as a tension sensor to regulate sarcomere assembly.
(A) Confocal micrographs of control muscles of a stage 17 wild-type embryo (left panels) and age-comparable zasp null mutant muscles (right panels) stained for MHC (blue in merge), α-actinin (red in merge) and Zasp (green in merge). Scale bar: 10 µm. (B) Confocal micrographs of control muscles of a stage 17 wild-type embryo (left panels) and same stage α-actinin null mutant muscles from Actn14 (right panels) stained for actin (blue in merge), α-actinin (red in merge) and MHC (green in merge). Scale bar: 10 µm. (C) Fluorescent confocal micrographs of control muscles of a stage 17 wild-type embryo (top panels) and α-actinin null mutant muscles from Actn14 of same stage (bottom panels) stained for Zasp (blue in merge), α-actinin (red in merge) and kettin (green in merge). Note that α-actinin null mutant muscles still have striated sarcomeres, but with expanded Z lines. Scale bar: 10 µm. (D) Primary muscle cells were treated with combinations of dsRNAs targeting components of the zipper/Zasp/α-actinin complex. Muscle striation was evaluated using anti-MHC, anti-actin and anti-α-actinin antibodies. Scale bar: 10 µm.
Figure 5.
Integrin is essential for sarcomere assembly and model.
(A) Fluorescent confocal micrographs of stage 17 wild-type muscles (left panels) and mys null mutant muscles of the same age (right panels) stained for actin (blue in merge), α-actinin (red in merge) and MHC (green in merge). DAPI staining reveals the multinucleated mys null mutant muscles. Note that I-Z-I collapsed to the center of muscles in the mys mutant, where thick filaments were mostly excluded. Scale bar: 10 µm. (B) A “two-state sarcomere assembly” model. Prior to sarcomere formation, various complexes, including integrin, tension sensor, I-Z-I complex, MHC filament and Tns-Tm, are assembled independently. Subsequently, the various complexes assemble and interact with the integrin pathway responsible for sarcomere stretching. Removal of any one of these complexes leads to a collapse and disorganization of the entire system. (C) Relationships between the sarcomeric functional complexes. The arrows indicate the interaction among these complexes as determined by the results presented in this study.