Article Figures & Data
Figures
- Figure 1.
Identification of Coronin 6 as a muscle-specific coronin family member. A, Schematic diagram of Coronin 6 domains showing the N-terminal extension (NE), seven-bladed β-propeller domain comprising 5 canonical WD40 repeats (WD), conserved C-terminal extension (CE), unique region (U), and coiled-coil domain (CC). B, Protein sequence alignment of rat Coronin 6 (rCoro6) with rat Coronin 1A (rCoro1A) generated by Clustal Omega. Solid black boxes represent the five predicted WD40 repeats, and dashed boxes represent two “hidden” repeats. C, Percentages of amino acid identities of Coronin 6 among different species. D, Percentages of amino acid identities between rCoro6 and other rat coronin proteins. E, Phylogenetic tree of mammalian coronin family generated by Clustal Omega. F, The tissue expression pattern of mammalian coronins in adult rats was revealed by northern blotting.
- Figure 2.
High concentrations of Coronin 6 at the NMJ. A, B, Specificity of Coronin 6 polyclonal antibody. Rabbit Coronin 6 polyclonal antibody recognized both the overexpressed Coronin 6 (Coro6) and endogenous Coronin 6 in adult rat muscle (Muscle) (A, left). This recognition was blocked by preincubation with the peptide antigen (A, right). Western blot analysis of COS-7 cells with ectopic expression of His-tagged Coro1C and Coro6 using the Coronin 6 polyclonal antibody (B). C, Coronin 6 protein expression in rat muscle during development was determined by Western blotting. D, Western blot analyses of Coronin 6 (20 μg per lane) were performed using rat gastrocnemius muscle after sciatic nerve crushing. Actin served as a loading control. E, C2C12 myoblasts in growth media (GM) or myotubes in differentiation media for the indicated times (h) were lysed and subjected to Western blotting to examine Coronin 6 protein expression. F, Coronin 6 was concentrated at the motor endplates during development (postnatal stages, from P1 to P21). Rat skeletal muscle sections were costained with antibody against Coronin 6 and AlexaFluor-555-conjugated α-BTX to visualize AChRs. Scale bar, 10 μm. Arrowheads indicate Coronin 6 staining colocalized with AChRs; arrows indicate Coronin 6 not colocalized with AChRs. G, Coronin 6 was localized at the postsynaptic muscle membrane of gastrocnemius muscle after nerve denervation. Scale bar, 10 μm. H, The immunoreactivity of Coronin 6 antibody was abolished by preabsorption with its peptide antigen. Scale bar, 10 μm.
- Figure 3.
Regulation of AChR clustering in myotubes by Coronin 6. A, C2C12 myotubes were transfected with siRNA against Coronin 6 (siCoro6) or luciferase as a control (siCont). Cell lysates were subjected to immunoblotting with antibodies against Coronin 6 or α-tubulin. B–D, Coronin 6 regulates agrin-induced AChR clustering. C2C12 myotubes were transfected with siCoro6 or siCont followed by stimulation with agrin for 12 h to induce AChR clustering. For AChR dispersal, AChR clusters on agrin-treated myotubes were labeled with α-BTX. Myotubes were subsequently washed and maintained in agrin-free medium for an additional 1, 4, or 12 h. Representative images (B), and quantification of the size (C) and number (D) of AChR clusters on myotubes from each condition. The mean ± SEM of at least 3 experiments is indicated. *p < 0.05, siCoro6 versus siCont (Student's t test). **p < 0.01, siCoro6 versus siCont (Student's t test). Scale bar, 50 μm. E, The percentage of AChR clusters was calculated by normalizing the number of AChR clusters at different time points after agrin withdrawal to that at baseline. *p < 0.05, percentage of AChR clusters at the first hour of agrin withdrawal versus that at baseline in Coronin 6-silenced myotubes. n.s., Not significant. F, C2C12 myoblasts were cultured on laminin-coated plates and fused for 2 d, and the myotubes were subsequently transfected with siCoro6 or siCont. AChR clusters were visualized by AlexaFluor-555-conjugated α-BTX. Three types of AChR clusters are shown: plaque, perforated, and branched clusters. G, The numbers of each type of AChR cluster were counted from at least three individual experiments (branched clusters: siCont, 23.4 ± 3.4; siCoro6, 13.6 ± 1.6). p = 0.033, siCoro6 versus siCont (Student's t test).
- Figure 4.
Association of Coronin 6 with AChR–actin complex upon agrin stimulation. A, Tyrosine phosphorylation level of AChRβ subunit in Coronin 6-knockdown myotubes. Coronin 6-knockdown myotubes were treated overnight with agrin, and cells were washed and incubated with agrin-free medium for 4 h. AChRs were precipitated with biotin-labeled α-BTX (Tox-P) and subsequently subjected to Western blot analysis using anti-4G10 antibody (p-Tyr-AChRβ). AChRβ was used as a loading control. B, Quantification of the fold change of p-Tyr-AChRβ levels (normalized to AChRβ) from three individual experiments. *p < 0.05, siCoro6 versus siCont (Student's t test). **p < 0.01, siCoro6 versus siCont (Student's t test). C, AChR extractability assay shows the linkage of AChR to the cytoskeleton after Coronin 6 knockdown. Myotubes were subjected to sequential extraction using different concentrations of Triton X-100. AChRs in the two fractions were precipitated by Tox-P, followed by Western blot analysis for the AChRβ subunit. D, Percentages of AChR β-subunit extracted from the 1% Triton X-100 fraction. Quantification was performed in three individual experiments. *p < 0.05, siCoro6 versus siCont (Student's t test). E, Actin cosedimentation assay. Coronin 6 and F-actin coprecipitated prominently in the pellet fraction but not the supernatants. F, Coimmunoprecipitation demonstrates Coronin 6 interacts with actin in C2C12 myotubes. G, Agrin treatment stimulated the recruitment of Coronin 6 to AChR clusters together with actin. Myotubes were treated with agrin for the indicated time periods. AChRs were precipitated by Tox-P followed by Coronin 6 (Coro6), actin, and AChRα immunoblotting.
- Figure 5.
Requirement of the actin-binding activity of Coronin 6 for its association with AChR clusters. A, Schematic diagram of Coronin 6 and its mutants. B, Interactions between Coronin 6 and its deletion and point mutants with actin in HEK293T cells. FLAG-tagged Coronin 6 and its mutants were overexpressed in HEK293T cells, and the protein was coimmunoprecipitated with anti-FLAG antibody. C-terminal FLAG-tagged Coronin 6 (WT; full-length [FL]), ΔCC mutant, ΔUCC mutant, or R29A/D mutant. C, D, The deletion of the coiled-coil domain or R29A/D mutants disrupted the association between Coronin 6 and AChRs. Myotubes were transfected with the mRNA of the Coronin 6 and its mutants. Cell lysates were subjected to biotin-conjugated α-BTX (Tox-P) for the precipitation of AChRs followed by Western blotting for FLAG. E, F, The cytoskeletal linkage of AChRs was reduced in myotubes overexpressing F-actin-binding-deficient Coronin 6 mutants. Myotubes were transfected with WT or mutant Coronin 6 mRNA and subsequently treated with agrin to induce AChR clusters. AChRs were then sequentially extracted by low and high concentrations of detergent as described in Materials and Methods. F, Percentages of AChRβ subunit extracted from the 1% Triton X-100 fraction. Quantification was performed in three individual experiments. *p < 0.05 R29D versus Control or WT (Student's t test). G–I, Overexpression of the Coronin 6 ΔUCC mutant significantly attenuated agrin-induced AChR clustering. Representative images (G), and quantification of the size (H) and number (I) of AChR clusters on myotubes from each condition. The mean ± SEM of at least 3 experiments is indicated. *p < 0.05, ΔUCC versus Control (Student's t test). Scale bar, 50 μm.
- Figure 6.
Coronin 6 knockdown perturbed AChR clustering in vivo. A, HEK-293T cells were cotransfected with Coronin 6 and pSUPER (−) or shCoro6 (+). Cell lysates were subjected to immunoblotting with antibodies against Coronin 6 or actin. B, The tibialis anterior muscles of adult mice were injected with 5 μg GFP and 30 μg pSUPER or shCoro6 followed by electroporation. Three weeks later, the muscles were stained with AlexaFluor-555-conjugated α-BTX to visualize AChR clusters. GFP signals indicate the transfected skeletal muscle fibers. Arrows indicate the normal pretzel-like structures of the NMJ. Impaired NMJ structures, such as fragmented (arrowhead) and circular patch-like structures (*), were observed in Coronin 6-knockdown fibers. C, Percentages of NMJs exhibiting pretzel-like, fragmented, and circular patch-like shapes (n = 31 from 4 mice injected with pSUPER; n = 36 from 4 mice injected with shCoro6). D, Quantification of the size of AChR clusters. **p < 0.01, shCoro6 versus pSUPER (Student's t test). E, Clustering of nuclei at the subsynaptic regions was unaltered in Coronin 6-silenced muscle. Nuclei and AChR clusters were visualized by DAPI (blue) and AlexaFluor-555-conjugated α-BTX staining (red), respectively. The subsynaptic regions are highlighted in rectangles. Scale bars, 20 μm.
