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First published online 27 May 2008
doi: 10.1242/jcs.021634

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Plectin 1 links intermediate filaments to costameric sarcolemma through β-synemin, -dystrobrevin and actin

¹ Department of Anatomy and Cell Biology, Faculty of Pharmacy, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo 202-8585, Japan
² Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan
³ Department of Anatomy, Nihon University School of Dentistry, Tokyo 101-8310, Japan
⁴ Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo 187-8502, Japan
⁵ Department of Anatomy, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan

View larger version (125K): [in this window] [in a new window]   Fig. 1. (A-R) Colocalization of β-synemin with plectin and -dystrobrevin at the costameric sarcolemma. β-Synemin colocalized precisely with plectin around Z-discs and beneath the sarcolemma (A-F), whereas it colocalized with -dystrobrevin beneath the sarcolemma (G-O), especially at costameric regions (L,O, arrows). Plectin also colocalized with -dystrobrevin at costameres (P-R, arrows in R). Bars, 5 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Immunoelectron microscopic analyses indicate the localization of β-synemin along IFs and in costameres. (A,B) Gold particles labeling β-synemin (arrowheads) were distributed along IFs and on subsarcolemmal dense plaques overlying Z-lines, or costameres. (C) A thin thread (arrow), possibly plectin molecules, projects from IF to a dense plaque labeled with gold particles. Bars, 0.1 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Mapping of the interacting sites between N-terminal plectin 1 (PleN1) fragments and full-length β-synemin by in vitro pull-down assay. (A) Schematic representations of the domain structures of full-length β-synemin and GST-fused recombinant β-synemin fragments used in the pull-down assay. The in vitro PleN1-binding phenotypes of mutant β-synemin fragments are summarized as + (strong binding) or ± (weak binding). GST-fused β-synemin fragments were incubated with Myc-tagged PleN1 and immunoprecipitated by anti-Myc antibody and protein L-agarose. These immunoprecipitates were subjected to immunoblotting with detection by anti-GST antibody. Each lane contained equivalent amounts of the immunoprecipitate, as represented by immunoblots of PleN1. (B) Domain structures of PleN1 and Myc-tagged recombinant plectin fragments, together with a summary of in vitro binding ability of plectin fragments to β-synemin tail fragments (Tail N1). Plectin fragments were incubated with GST-fused Tail N1 and pulled down using glutathione beads, followed by immunoblotting with anti-Myc antibody. Each lane contained equivalent amounts of the immunoprecipitate, as represented by immunoblots of Tail N1.

View larger version (116K): [in this window] [in a new window]   Fig. 4. Subcellular localization of mutant plectin fragments containing a β-synemin-binding site in transfected C2C12 myoblasts and myotubes. C2C12 myoblasts were transfected with plasmids expressing Myc-tagged plectin Ex 1, CHD and PlD-M fragments, and then cultured in differentiation medium. At 60-84 hours post transfection, differentiated myotubes were immunolabeled with antibodies specific for Myc and β-synemin. Localization of β-synemin along SFLSs and throughout the sarcoplasm in a dotted pattern was found in control myotubes doubly immunolabeled with Alxea-Fluor-594-conjugated phallotoxins and anti-β-synemin pAb (A-F). The three mutant plectin fragments more or less colocalized with β-synemin along SFLS (arrows in J-L,P-R,V-X). Arrowheads in V and X indicate sites associated predominantly with β-synemin, but scarcely with PlD-M. Bars, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 5. In vitro interactions of plectin 1 and β-synemin with F-actin. Bindings of plectin CHD and β-synemin Tail N1 fragments to F-actin were demonstrated by actin co-sedimentation assay. After centrifugation, most of CHD fragments (3 µM) were found in the pellet (P) in the presence of F-actin (6 µM), whereas they remained in the supernatant (S) in the absence of actin (lanes 2 and 3). Small amounts of β-synemin Tail N1 fragments were sedimented in the presence of F-actin (15 µM and 30 µM β-synemin in lanes 4 and 5, respectively), while in the absence of F-actin, the Tail N1 fragments (30 µM) were observed mostly in the supernatant (lanes 6). Even in the presence of a 10-fold molar excess of Tail N1 fragments (30 µM), CHD fragments were still found mostly in the pellet together with F-actin (compare lanes 7 with lanes 8).

View larger version (18K): [in this window] [in a new window]   Fig. 6. In vitro interactions of plectin 1 with -dystrobrevin and β-synemin. (A) -Dystrobrevin fragments (-Dbr, 0.8 µg) were incubated with GST-fused β-synemin (β-Syn, 10 µg) or GST (1.8 µg) in the incubation buffer (1 ml) and then precipitated by glutathione beads (Gl-beads, 50 µl). GST-fused β-Syn precipitated -Dbr, but GST alone did not. (B) -Dbr (0.5 µg) were incubated with Myc-tagged plectin 1 (PleN1, 9.6 µg) or plectin 1f fragments (PleN1f, 3.2 µg) or β-galactosidase (LacZ, 7.6 µg) in the incubation buffer (1.4 ml) including 0.4% BSA and then immunoprecipitated with anti-Myc antibody (4 µg) and protein L-agarose (40 µl). PleN1 coimmunoprecipitated with -Dbr, but neither PleN1f nor LacZ did. (C) -Dbr (0.4 µg) and GST-fused β-Syn (2 µg)were incubated with Myc-tagged PleN1 (9 µg) or PleN1f (3 µg) or LacZ (9 µg) in the incubation buffer (1.2 ml) including 0.4% BSA and then immunoprecipitated with anti-Myc antibody (4 µg) and protein L-agarose (40 µl). PleN1 coimmunoprecipitated with both β-Syn and -Dbr, whereas PleN1f pulled down only β-Syn. However, LacZ and PleN1 in combination with control IgG pulled down none of them.

View larger version (20K): [in this window] [in a new window]   Fig. 7. The assessment of the formation of plectin 1 complexes with F-actin, -dystrobrevin and β-synemin by blot overlay assays. PleN1 fragments (1 µg) immobilized on nitrocellulose were incubated with 3 µM F-actin (A) or 1 µM -dystrobrevin fragments (D) or 1 µM GST-fused full-length β-synemin (S) or the mixture of the three proteins (M), followed by detection of bound proteins using antibody specific for each protein. The signals resulting from the overlay with the three proteins were almost equivalent to those from the overlay with each protein alone. Values represent the mean ± s.d. (error bars) of two spots in three independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 8. In vivo interactions of plectin and β-synemin with costameric proteins including DGC. Plectin- and β-synemin-immune complexes were immunoprecipitated from LM lysates by anti-plectin or anti-β-synemin antibodies, respectively. Both the immune complexes contained dystrophin, -dystrobrevin 1 and -dystrobrevin 2, meta-vinculin, desmin and actin, including nonmuscle actin, but not -actinin. In control experiments, control IgG did not significantly coimmunoprecipitate with any proteins.

View larger version (45K): [in this window] [in a new window]   Fig. 9. Immunoblots of costameric, IF-associated and IF proteins from wild-type (N) and dystrophin-deficient (A) mdx mice (5-6 weeks old) and CXMDJ dogs (6-10 months old). (A,B) Reduced expression of -dystrobrevin 1, -dystrobrevin 2 and -dystrobrevin 3, and increased expression of utrophin, integrin β1d, metavinculin and vimentin were detected in dystrophin-deficient mice and dogs when compared with wild-type controls. Note slightly elevated expression of plectin and β-synemin in dystrophin-deficient dogs. Values represent the mean ± s.d. (error bars) of muscle specimens obtained from three mice and dogs. **P<0.01 and *P<0.05, compared with control mice or dogs.

View larger version (16K): [in this window] [in a new window]   Fig. 10. Models of molecular organization from IF to costameres including DGC and integrin complexes. (A) The N-terminal part of plectin 1 is associated with costameres by binding β-synemin, costameric actin and -dystrobrevin, whereas its C-terminal part is linking to IFs. Exon 1 part of plectin 1 might interact with not only -dystrobrevin but also β-synemin, whereas β-synemin on plectin molecules might be further associated with costameric actin linking to plectin, dystrophin and metavinculin. (B) As an alternative model, direct interactions of plectin with dystrophin and β-dystroglycan were proposed by Rezniczek et al. (Rezniczek et al., 2007). This model is partly modified to represent the interaction of plectin 1 with -dystrobrevin through its exon 1 part.

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