Functional Analysis of the Xin-Repeat Protein Family in Cross-striated Muscle

The assembly of the sarcomere of cross-striated muscle involves a plethora of transcription factors, signalling and structural proteins and requires their coordinate interplay in order to construct this uniquely regular structure. Its early upregulation in muscle precursor cells during embryonic development and in activated satellite cells upon skeletal muscle damage render the protein Xin an interesting candidate for being involved in orchestrating muscle morphogenesis. The establishment of the H-2K cell line as a new reliable model system for studying myofibrillogenesis in vitro using immunofluorescence microscopy and protein expression analysis of major structural components of the sarcomere in this work provided a new tool to investigate Xin function during myogenic differentiation. Examining the transcription level revealed that all three Xin isoforms A, B and C are being upregulated upon initiation of differentiation while downregulated as soon as H-2K cells gain their contractility. On the protein level, only the expression of isoforms A and B was doubtlessly identified exhibiting a course related to their transcriptional level. During differentiation, Xin was strongly associated with non-striated myofibrils (NSMF), where it colocalized with its binding partner filamin C, but not with Z-disc precursor structures. Consistently, in contractile myotubes Xin was absent from mature Z-discs but longitudinally connected to immature sarcomeres. In this work, the SH3 domains of nebulin and nebulette were identified as novel ligands of Xin isoforms A and C and peptide scans elucidated for the first time the exact peptide motif and the residues essential for this interaction. Bimolecular fluorescence complementation (BiFC) assays of Xin C and nebulette in embryonic cardiomyocytes selectively demonstrated the potential cellular sites of this interaction since BiFC complexes were exclusively formed along NSMF’s in early developmental stages but not at mature Z-discs. This finding was corroborated by localization studies of nebulin and Xin in H-2K cells as colocalization was only observed at specific sites of NSMF’s substantiating the developmental regulation of this interaction. Identification of the protein LIMCH1 as the first binding partner exclusively interacting with the largest Xin isoform A provided first insights into isoform-specific functions. Yeast two-hybrid and biochemical studies mapped the binding interface to the C-terminal LIM domain of LIMCH1 and impressively showed its specificity. Investigation on the mRNA and protein level during myofibrillogenesis of H-2K cells displayed that LIMCH1 is also upregulated at the onset of differentiation constantly increasing until the final stage pointing to an interaction during that process. In rat skeletal muscle tissue and H-2K cells an additional larger LIMCH1 isoform (LIMCH1 muscle) was detected which pre-