Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling

Abstract

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

FIGURE 1:

Schematic representation of domain structures of Xin isoforms and nebulin. (A) Molecular layout of the three known Xin variants and the bait construct used for the yeast two-hybrid screen (Xin PR3+4). (B) Domain organization of nebulin, together with some prey constructs (dark green bars) from the yeast two-hybrid screen. The smallest interacting construct comprises the SH3 domain and is part of the serine-rich linker. Domains are explained in the appendant boxes.

FIGURE 2:

Summary of yeast two-hybrid analysis of interaction with Xin-repeat proteins. Combinations of baits and preys examined in yeast two-hybrid assays and the experimental results (+, –, interaction and no interaction, respectively). Note that proline-rich regions of both Xin and XIRP2 bind to the carboxy terminus of nebulin and nebulette.

FIGURE 3:

Biochemical verification of the interaction of Xin-repeat proteins with nebulin and nebulette in coimmunoprecipitation (coIP) assays. Recombinant fragments of nebulin (NEB m180) and nebulette (NET SH3+L) were mixed with fragments of Xin (PR4) or XIRP2 (PR2-6) as indicated at the top of each lane and precipitated with (+) or without (–) the T7 mAb (blot: IP). Binding of Xin (A, C) or XIRP2 (B, D) fragments was demonstrated by staining duplicate blots with an antibody against their respective tags (myc tag or EEF tag) marked with coIP. All fragments of Xin and XIRP2 were coprecipitated both with nebulin and nebulette fragments. Controls without antibody and/or interaction partner confirmed specific binding. Numbers at the left indicate positions of molecular mass markers in kilodaltons, and arrows show the positions of the individual constructs.

FIGURE 4:

Peptide array analysis to identify the peptide motifs in Xin and XIRP2 mediating binding to the SH3 domain of nebulette. Arrays of overlapping peptides representing binding regions of Xin (A) and XIRP2 (B) were synthesized on a modified cellulose membrane and overlaid with a GST-tagged SH3 domain of nebulette. Peptides 80 and 81 of Xin and 50 and 51 of XIRP2 showed the strongest specific binding to the SH3 domain. Additional signals were observed with only GST or only secondary antibody and therefore are unspecific. Respective peptide sequences are aligned and the overlapping amino acids boxed. Right, the complete substitutional analysis, in which each amino acid of the interacting peptide (wt; y-axis) was exchanged for any other amino acid (spotted along the x-axis). Both nebulette SH3 domain–binding peptides in Xin and XIRP2 contain a proline-rich motif and a positively charged lysine residue flanking this sequence C-terminally. The established overall consensus motif therefore is PPXXXPKP, with as minimal consensus PXXXPK.

FIGURE 5:

Ribbon/stick presentation of the nebulin SH3–XIRP2 peptide complex. The nebulin SH3 domain is in orange, and the XIRP2 peptide is in cyan. All visible XIRP2 residues and those nebulin SH3 domain residues that are involved in specific interactions with XIRP2 are labeled. Intermolecular hydrogen bonds are shown by dashed lines.

FIGURE 6:

Immunolocalization of Xin and nebulin in differentiating mouse myocytes. Confocal laser scanning microscopy images of H-2K cells at different developmental stages stained with antibodies against Xin (A, E, H, L, O) and nebulin (B, F, I, M, P). In merged images (C, D, G, J, K, N, Q) Xin and nebulin are tinted green and red, respectively. Boxed areas in A–D and K are shown enlarged in insets (A–C) or in neighboring pictures (E–J, L–Q). In early-differentiating cells, Xin and nebulin partially colocalize along actin filament bundles (A–C, arrows; insets, arrowheads). More extensive colocalization is observed in nascent myofibrils (D; H–J, arrowheads). Xin weakly labels early myofibrils in a punctate pattern during developmental stages in which nebulin starts to be organized in Z-bodies (E–G, arrows). The colocalization seems to be lost on assembly of mature Z-discs that contain only nebulin (K–Q). In spontaneously contracting cells with cross-striated myofibrils (K–Q), Xin and nebulin colocalize in longitudinally oriented fibrillar structures that represent immature myofibrils or areas of remodeling (L–Q, arrowheads), whereas only nebulin is localized in Z-discs. Bar, 10 μm.

FIGURE 7:

Transfection of embryonic mouse cardiomyocytes with XinC- and nebulette-encoding cDNAs. Venus-tagged nebulette targeted to nonstriated premyofibrils (arrow), whereas it colocalized with a Z-disc titin epitope (arrowheads) in regions exhibiting myofibrils (A–C). In contrast, XinC was prominently localized along premyofibrils, as indicated by staining of Xin (D–F), but not to Z-discs in further-differentiated cardiomyocytes. Note that the antibody used to stain Xin recognizes XinA and B but not XinC. In myofibrillar areas Xin specifically targeted to longitudinal strands between adjacent Z-discs identified by titin staining (G–I). Cotransfection of XinC and nebulette coupled to complementary nonfluorescent Venus fragments resulted in BiFC complex formation at premyofibrils of undifferentiated cells (J–L). In differentiated cardiomyocytes this complex became restricted to longitudinal strands between adjacent Z-discs (M–O). Bar, 10 μm.

FIGURE 8:

BiFC analysis of the interaction between XinB+PR4 and nebulette in C2C12 myocytes. BiFC complexes of Venus1-nebulette and XinB+PR4-Venus2 continuously decorate membrane-associated premyofibrils at early differentiation stages (A–C, arrows). In cells showing premyofibrils and nascent myofibrils with Z-bodies (D–I), the BiFC complex is continuously associated with these structures (G–I, arrows and open arrowheads, respectively). After the formation of striated myofibrils the complex is restricted to early Z-discs (G–I, closed arrowheads), whereas no signal is visible in mature Z-discs. (G–I) Magnification of the boxed areas in D–F. An antibody against a Z-disc epitope of titin was used to localize Z-discs. Bar, 10 μm.

FIGURE 9:

Immunolocalization of Xin, XIRP2, nebulin, and the Z-disc marker filamin C in mouse skeletal muscle sections. Sections were stained for Xin, XIRP2, and N2-line nebulin (A, B) or Xin, filamin C, and Z-disc nebulin, respectively (C). Xin is exclusively detected together with XIRP2 and filamin C along longitudinal strands between individual Z-discs. These areas of minor muscle damage show diffuse nebulin localization (A–C) that partially overlaps with Xin and filamin C (C, arrows). These regions often contain supernumerary sarcomeres, as indicated by the filamin C signal (compare asterisks and arrowheads in C). Bar, 20 μm (A), 5 μm (B, C).