Search

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.

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.

Mechanical force-induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.

Filamin c (FLNc) is a large dimeric actin-binding protein located at premyofibrils, myofibrillar Z-discs and myofibrillar attachment sites of striated muscle cells, where it is involved in mechanical stabilization, mechanosensation and intracellular signaling. Mutations in the gene encoding FLNc give rise to skeletal muscle diseases and cardiomyopathies. Here, we demonstrate by fluorescence recovery after photobleaching that a large fraction of FLNc is highly mobile in cultured neonatal mouse cardiomyocytes and in cardiac and skeletal muscles of live transgenic zebrafish embryos. Analysis of cardiomyocytes from Xirp1 and Xirp2 deficient animals indicates that both Xin actin-binding repeat-containing proteins stabilize FLNc selectively in premyofibrils. Using a novel assay to analyze myofibrillar microdamage and subsequent repair in cultured contracting cardiomyocytes by live cell imaging, we demonstrate that repair of damaged myofibrils is achieved within only 4 h, even in the absence of de novo protein synthesis. FLNc is immediately recruited to these sarcomeric lesions together with its binding partner aciculin and precedes detectable assembly of filamentous actin and recruitment of other myofibrillar proteins. These data disclose an unprecedented degree of flexibility of the almost crystalline contractile machinery and imply FLNc as a dynamic signaling hub, rather than a primarily structural protein. Our myofibrillar damage/repair model illustrates how (cardio)myocytes are kept functional in their mechanically and metabolically strained environment. Our results help to better understand the pathomechanisms and pathophysiology of early stages of FLNc-related myofibrillar myopathy and skeletal and cardiac diseases preceding pathological protein aggregation., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2014. Published by The Company of Biologists Ltd.)

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. [ABSTRACT FROM AUTHOR]