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Recent studies have shown that certain types of transformed cells are extruded from an epithelial monolayer. However, it is not known whether and how neighbouring normal cells play an active role in this process. In this study, we demonstrate that filamin A and vimentin accumulate in normal cells specifically at the interface with Src- or RasV12-transformed cells. Knockdown of filamin A or vimentin in normal cells profoundly suppresses apical extrusion of the neighbouring transformed cells. In addition, we show in zebrafish embryos that filamin plays a positive role in the elimination of the transformed cells. Furthermore, the Rho/Rho kinase pathway regulates filamin accumulation and filamin acts upstream of vimentin in the apical extrusion. This is the first report demonstrating that normal epithelial cells recognize and actively eliminate neighbouring transformed cells and that filamin is a key mediator in the interaction between normal and transformed epithelial cells. Mutations resulting in cell transformation occur in single cells to begin with and these cells can be eliminated from epithelial layers. Here, the authors describe a mechanism whereby neighbouring normal cells mediate this extrusion using filamin and intermediate filament, vimentin.

We investigated the interaction of HspB7 and its α-crystallin domain with the wild-type (WT) C-terminal fragment of human filamin C (FLNC), containing immunoglobulin-like domains 22–24 and its three mutants associated with cardio- and myopathies. The physicochemical properties of the WT FLNC fragment and its three mutants, p.Glu2472_Asn2473delinsAsp (EN/D) located in the 22nd domain, p.P2643_L2645del (ΔPGL), and p.W2710X (Wmut) both located in the 24th immunoglobulin-like domain were analyzed. Although all FLNC fragments had similar secondary structures, WT FLNC and its EN/D and ΔPGL mutants formed dimers, whereas Wmut formed either monomers or aggregates. The surface hydrophobicity of EN/D, ΔPGL, and especially Wmut mutants was larger than that of the WT fragment. Size exclusion chromatography, native gel electrophoresis, and chemical crosslinking indicated that the efficiency of interaction with HspB7 or its α-crystallin domain decreased in the order WT~EN/D > ΔPGL. Wmut was unable to interact with either HspB7 or its α-crystallin domain. Modeling via Alphafold 3 indicated that EN/D mutation affected the orientation of two loops connecting β-strands in the 22nd domain, while the ΔPGL and Wmut mutations exposed a hydrophobic groove in the 24th domain thereby reducing their interaction with HspB7. These findings reveal the molecular mechanisms underlying filaminopathies associated with three mutations in the C-terminal region of filamin C.

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.

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (FlncAGA/AGA and FlncGA/GA) were embryonically lethal. We determined that FlncGA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (FlncGA/wt, FlncAGA/wt, and FlncGA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (FlncGA/wt, FlncAGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (FlncGA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, FlncGA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

Cardiomyopathy affects approximately 1 in 500 adults and is the leading cause of death. Familial cases are common, and mutations in many genes are involved in cardiomyopathy, especially those in genes encoding cytoskeletal, sarcomere, and nuclear envelope proteins. Filamin C is an actin-binding protein encoded by filamin C (FLNC) gene and participates in sarcomere stability maintenance. FLNC was first demonstrated to be a causal gene of myofibrillar myopathy; recently, it has been found that FLNC mutation plays a critical role in the pathogenesis of cardiomyopathy. In this review, we summarized the physiological roles of filamin C in cardiomyocytes and the genetic evidence for links between FLNC mutations and cardiomyopathies. Truncated FLNC is enriched in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. Non-truncated FLNC is enriched in hypertrophic cardiomyopathy and restrictive cardiomyopathy. Two major pathomechanisms in FLNC-related cardiomyopathy have been described: protein aggregation resulting from non-truncating mutations and haploinsufficiency triggered by filamin C truncation. Therefore, it is important to understand the cellular biology and molecular regulation of FLNC to design new therapies to treat patients with FLNC-related cardiomyopathy.

The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation. Filamin C is a key actin-binding protein involved in cardiomyopathies and musculoskeletal disorders. Here, Wang et al reveal that it interacts with the heat shock protein HSPB7 under biomechanical stress, forming a stable hetero-dimer which is regulated by phosphorylation.

Augmenting the portfolio of therapeutics for type 2-driven diseases is crucial to address unmet clinical needs and to design personalized treatment schemes. An attractive therapy for such diseases would consist in targeting the recruitment of T helper 2 (Th2) lymphocytes to inflammatory sites. Herein, we show the degradation of filamins (FLN) a and b by the ASB2α E3 ubiquitin ligase as a mechanism sustaining Th2 lymphocyte functions. Low levels of FLNa and FLNb confer an elongated shape to Th2 lymphocytes associated with efficient α V β 3 integrin-dependent cell migration. Genes encoding the α V β 3 integrin and ASB2α belong to the core of Th2-specific genes. Using genetically modified mice, we find that increasing the levels of FLNa and FLNb in Th2 lymphocytes reduces airway inflammation through diminished Th2 lymphocyte recruitment in inflamed lungs. Collectively, our results highlight ASB2α and its substrates FLNa and FLNb to alter Th2 lymphocyte-mediated responses. Filamin proteins are degraded by ASB2α E3 ubiquitin ligase and may affect T cell function. Here the authors show that increased levels of Filamin A and Filamin B in Th2 cells reduces allergic inflammation in mouse models through reduced Th2 cell recruitment into inflamed lungs.

Early detection improves survival and increases curative probability in hepatocellular carcinoma (HCC). Peripheral blood mononuclear cells (PBMCs) can provide an inexpensive, less-invasive and highly accurate method. The objective of this study is to find the potential marker for HCC screening, utilizing gene expression of the PBMCs. Data from the NCBI GEO database of gene expression in HCC patients and healthy donor's PBMCs was collected. As a result, GSE 49515 and GSE 58208 were found. Using both, a statistical significance test was conducted in each gene expression of each data set which resulted in 187 genes. We randomized three selected genes (FLNA, CAP1, and CLU) from the significant p -value group ( p -values < 0.001). Then, a total of 76 healthy donors, 153 HCC, 20 hepatic fibrosis, 20 non-alcoholic fatty liver were collected. Quantitative RT-PCR (qRT-PCR) was performed in cDNA from all blood samples from the qRT-PCR, The Cycle threshold (Ct) value of FLNA, CLU, CAP1 of HCC group (28.47 ± 4.43, 28.01 ± 3.75, 29.64 ± 3.90) were lower than healthy group (34.23 ± 3.54, 32.90 ± 4.15, 32.18 ± 5.02) ( p -values < 0.0001). The accuracy, sensitivity and specificity of these genes as a screening tool were: FLNA (80.8%, 88.0%, 65.8%), CLU (63.4%, 93.3%, 31.3%), CAP1 (67.2%, 83.3%, 39.1%). The tests were performed in two and three gene combinations. Results demonstrated high accuracy of 86.2%, sensitivity of 85% and specificity of 88.4% in the FLNA and CLU combination. Furthermore, after analyzed using hepatic fibrosis and non-alcoholic fatty liver as a control, the FLNA and CLU combination is shown to have accuracy of 76.9%, sensitivity of 77.6% and specificity of 75%. Also, we founded that our gene combination performs better than the current gold standard for HCC screening. We concluded that FLNA and CLU combination have high potential for being HCC novel markers. Combined with current tumor markers, further research of the gene’s expression might help identify more potential markers and improve diagnosis methods.

Acromegaly, caused by growth hormone-secreting pituitary tumors, often causes significant challenges in its management due to poor surgical outcomes and resistance to pharmacological treatment. The present study aims to explore the expression of Filamin A (FLNA), a cytoskeletal protein involved in somatostatin receptor signaling, and its clinical relevance in acromegaly. We conducted immunohistochemical (IHC) study on 34 GH-secreting pituitary tumors to evaluate FLNA expression intensity and its associations with somatostatin receptors (SSTR2, SSTR5), E-Cadherin, tumor characteristics obtained through imaging studies, and pharmacological treatment responses. Our findings revealed a 100% FLNA positivity rate, with moderate to strong FLNA expression correlating significantly with SSTR5 expression and the presence of suprasellar tumor extension, indicating a potential role in tumor invasiveness. Moreover, patients with macrodenomas presented significantly higher FLNA intensity compared to the ones with microadenomas. FLNA expression showed no significant association with SSTR2, E-Cadherin, surgical cure rate or first-generation somatostatin receptor ligand (fgSRL) responses. However, the series of patients treated with Pasireotide ( n  = 4) demonstrated a trend suggesting better biochemical control with higher FLNA expression. In conclusion, our results suggest that FLNA may be associated with tumor invasiveness in GH-secreting pituitary tumors. While data on Pasireotide-treated patients are exploratory, further studies are needed to assess FLNA’s potential as a treatment response marker in acromegaly.