Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, β-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of β-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither β-H-Spectrin nor Big bang require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H-33) displaces endogenous β-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.

Filamin A is an essential protein in the cell cytoskeleton because of its actin binding properties and unique homodimer rod-shaped structure, which organises actin into three-dimensional orthogonal networks imperative to cell motility, spreading and adhesion. Filamin A is subject to extensive posttranslational modification (PTM) which serves to co-ordinate cellular architecture and to modulate its large protein-protein interaction network which is key to the protein\'s role as a cellular signalling hub. Characterised PTMs include phosphorylation, irreversible cleavage, ubiquitin mediated degradation, hydroxylation and O-GlcNAcylation, with preliminary evidence of tyrosylation, carbonylation and acetylation. Each modification and its relation to filamin A function will be described here. These modifications are often aberrantly applied in a range of diseases including, but not limited to, cancer, cardiovascular disease and neurological disease and we discuss the concept of target specific PTMs with novel therapeutic modalities. In summary, our review represents a topical \'one-stop-shop\' that enables understanding of filamin A function in cell homeostasis and provides insight into how a variety of modifications add an extra level of Filamin A control.

BACKGROUND: Thoracic aortic aneurysm (TAA) is associated with significant morbidity and mortality. Although individuals with family histories of TAA often undergo clinical molecular genetic testing, adults with nonsyndromic TAA are not typically evaluated for genetic causes. We sought to understand the genetic contribution of both germline and somatic mosaic variants in a cohort of adult individuals with nonsyndromic TAA at a single center.
RESULTS: One hundred eighty-one consecutive patients <60 years who presented with nonsyndromic TAA at the Massachusetts General Hospital underwent deep (>500×) targeted sequencing across 114 candidate genes associated with TAA and its related functional pathways. Samples from 354 age- and sex-matched individuals without TAA were also sequenced, with a 2:1 matching. We found significant enrichments for germline (odds ratio [OR], 2.44, P=4.6×10-6 [95% CI, 1.67-3.58]) and also somatic mosaic variants (OR, 4.71, P=0.026 [95% CI, 1.20-18.43]) between individuals with and without TAA. Likely genetic causes were present in 24% with nonsyndromic TAA, of which 21% arose from germline variants and 3% from somatic mosaic alleles. The 3 most frequently mutated genes in our cohort were FLNA (encoding Filamin A), NOTCH3 (encoding Notch receptor 3), and FBN1 (encoding Fibrillin-1). There was increased frequency of both missense and loss of function variants in TAA individuals.
CONCLUSIONS: Likely contributory dominant acting genetic variants were found in almost one quarter of nonsyndromic adults with TAA. Our findings suggest a more extensive genetic architecture to TAA than expected and that genetic testing may improve the care and clinical management of adults with nonsyndromic TAA.

Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease associations have yet to be determined for the increasing number of genes included in clinical panels. Genetic variants evaluation was undertaken for the most relevant non-desmosomal disease genes. We retrospectively studied 320 unrelated Italian ACM patients, including 243 cases with predominant right-ventricular (ARVC) and 77 cases with predominant left-ventricular (ALVC) involvement, who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes. The aim was to assess rare genetic variants in transmembrane protein 43 (TMEM43), desmin (DES), phospholamban (PLN), filamin c (FLNC), cadherin 2 (CDH2), and tight junction protein 1 (TJP1), based on current adjudication guidelines and reappraisal on reported literature data. Thirty-five rare genetic variants, including 23 (64%) P/LP, were identified in 39 patients (16/243 ARVC; 23/77 ALVC): 22 FLNC, 9 DES, 2 TMEM43, and 2 CDH2. No P/LP variants were found in PLN and TJP1 genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment for TMEM43 (3.79-fold), DES (10.31-fold), PLN (117.8-fold) and FLNC (107-fold). A non-desmosomal rare genetic variant is found in a minority of ARVC patients but in about one third of ALVC patients; as such, clinical decision-making should be driven by genes with robust evidence. More than two thirds of non-desmosomal P/LP variants occur in FLNC.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) remains a deadly disease with a poor prognosis. Here, we identified the ETS homologous factor (EHF) and its target Filamin-B (FLNB) as molecules related to immune evasion in ccRCC. We also explored the upstream modifier that manipulates EHF in ccRCC.
METHODS: Cell proliferation and apoptosis assay, wound healing assay, and Transwell assay were designed to analyze the effects of EHF or FLNB knockdown on the biological activity of ccRCC cells. The growth of differently treated ccRCC cells was assessed by orthotopic tumors. ccRCC cells with different treatments were co-cultured with macrophages, and the role of the lysine-specific demethylase 5B (KDM5B)/EHF/FLNB axis on macrophage polarization or ccRCC progression was characterized by detecting the expression of M2 macrophage markers in the co-culture system or tumor tissues of tumor-bearing mice.
RESULTS: The expression of EHF and FLNB was higher, while KDM5B was lower in HK2 cells than in ccRCC cells. EHF overexpression inhibited the biological behavior of ccRCC cells and tumor growth in mice. EHF activated FLNB transcription. Knockdown of FLNB supported the biological activity of ccRCC cells and tumor growth and reversed M2 macrophage polarization in tumor tissues of mice in the presence of EHF. KDM5B inhibited EHF expression by H3K4me3 demethylation, and EHF knockdown potentiated M2 macrophage polarization and tumor growth in vivo repressed by KDM5B knockdown.
CONCLUSIONS: KDM5B inhibited the expression of EHF by repressing H3K4me3 modification and the transcription of FLNB by EHF to promote immune evasion and progression of ccRCC.

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.

BACKGROUND: Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood.
RESULTS: We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib.
CONCLUSIONS: FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Mechanical cues from the tissue microenvironment, such as the stiffness of the extracellular matrix, modulate cellular forms and functions. As numerous studies have shown, this modulation depends on the stiffness-dependent remodeling of cytoskeletal elements. In contrast, very little is known about how the intracellular organelles such as mitochondria respond to matrix stiffness and whether their form, function, and localization change accordingly. Here, we performed an extensive quantitative characterization of mitochondrial morphology, subcellular localization, dynamics, and membrane tension on soft and stiff matrices. This characterization revealed that while matrix stiffness affected all these aspects, matrix stiffening most distinctively led to an increased perinuclear clustering of mitochondria. Subsequently, we could identify the matrix stiffness-sensitive perinuclear localization of filamin as the key factor dictating this perinuclear clustering. The perinuclear and peripheral mitochondrial populations differed in their motility on soft matrix but surprisingly they did not show any difference on stiff matrix. Finally, perinuclear mitochondrial clustering appeared to be crucial for the nuclear localization of RUNX2 and hence for priming human mesenchymal stem cells towards osteogenesis on a stiff matrix. Taken together, we elucidate a dependence of mitochondrial localization on matrix stiffness, which possibly enables a cell to adapt to its microenvironment.

OBJECTIVE: Genetic disorders involved in skeleton system arise due to the disturbance in skeletal development, growth and homeostasis. Filamin B is an actin binding protein which is large dimeric protein which cross link actin cytoskeleton filaments into dynamic structure. A single nucleotide changes in the FLNB gene causes spondylocarpotarsal synostosis syndrome, a rare bone disorder due to which the fusion of carpels and tarsals synostosis occurred along with fused vertebrae. In the current study we investigated a family residing in north-western areas of Pakistan.
METHODS: The whole exome sequencing of proband was performed followed by Sanger sequencing of all family members of the subject to validate the variant segregation within the family. Bioinformatics tools were utilized to assess the pathogenicity of the variant.
RESULTS: Whole Exome Sequencing revealed a novel variant (NM_001457: c.209C>T and p.Pro70Leu) in the FLNB gene which was homozygous missense mutation in the FLNB gene. The variant was further validated and visualized by Sanger sequencing and protein structure studies respectively as mentioned before.
CONCLUSIONS: The findings have highlighted the importance of the molecular diagnosis in SCT (spondylocarpotarsal synostosis syndrome) for genetic risk counselling in consanguineous families.