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Variations in the gene encoding filamin-A-interacting protein 1 (FILIP1) were identified to be associated with a combination of neurological and muscular symptoms. While FILIP1 was shown to regulate motility of brain ventricular zone cells, a process important for corticogenesis, the function of the protein in muscle cells has been less well characterized. The expression of FILIP1 in regenerating muscle fibres predicted a role in early muscle differentiation. Here we analysed expression and localization of FILIP1 and its binding partners filamin-C (FLNc) and microtubule plus-end-binding protein EB3 in differentiating cultured myotubes and adult skeletal muscle. Prior to the development of cross-striated myofibrils, FILIP1 is associated with microtubules and colocalizes with EB3. During further myofibril maturation its localization changes, and FILIP1 localizes to myofibrillar Z-discs together with the actin-binding protein FLNc. Forced contractions of myotubes by electrical pulse stimulation (EPS) induce focal disruptions in myofibrils and translocation of both proteins from Z-discs to these lesions, suggesting a role in induction and/or repair of these structures. The immediate proximity of tyrosylated, dynamic microtubules and EB3 to lesions implies that also these play a role in these processes. This implication is supported by the fact that in nocodazole-treated myotubes that lack functional microtubules, the number of lesions induced by EPS is significantly reduced. In summary, we here show that FILIP1 is a cytolinker protein that is associated with both microtubules and actin filaments, and might play a role in the assembly of myofibrils and their stabilization upon mechanical stress to protect them from damage. [ABSTRACT FROM AUTHOR]

Filamin C (FLNc) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adaptor proteins that are mainly expressed in cardiac and skeletal muscles and which play important roles in the assembly and repair of myofibrils and their attachment to the membrane. We identified the dystrophin-binding protein aciculin (also known as phosphoglucomutase-like protein 5, PGM5) as a new interaction partner of FLNc and Xin. All three proteins colocalized at intercalated discs of cardiac muscle and myotendinous junctions of skeletal muscle, whereas FLNc and aciculin also colocalized in mature Z-discs. Bimolecular fluorescence complementation experiments in developing cultured mammalian skeletal muscle cells demonstrated that Xin and aciculin also interact in FLNc-containing immature myofibrils and areas of myofibrillar remodeling and repair induced by electrical pulse stimulation (EPS). Fluorescence recovery after photobleaching (FRAP) experiments showed that aciculin is a highly dynamic and mobile protein. Aciculin knockdown in myotubes led to failure in myofibril assembly, alignment and membrane attachment, and a massive reduction in myofibril number. A highly similar phenotype was found upon depletion of aciculin in zebrafish embryos. Our results point to a thus far unappreciated, but essential, function of aciculin in myofibril formation, maintenance and remodeling. [ABSTRACT FROM AUTHOR]

Background: The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins. Methods: We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development. Results: Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as betatropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms ofpathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, anddisturbed assembly of cytoskeletal, myofibrillar structures and muscle development. Conclusions: In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicatesthat they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects inspecific sarcomeric constituents. [ABSTRACT FROM AUTHOR]

Filamin C (FLNc) is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familial and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from FLNc and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: in comparison to wildtype animals (i) the expression level of FLNc protein is drastically reduced; (ii) mutant FLNc is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack Bcl2-associated athanogene 3 (BAG3) protein; (iv) the expression of heat shock protein beta-7 (HSPB7) is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective FLNc dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative, more amino-terminally located dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that FLNc is important for mechanosensing in the context of Z-disc stabilization and maintenance. [ABSTRACT FROM AUTHOR]