Protein Filamin Research Articles

Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation.

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)–based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P < 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P < 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulates SST2 and SST5 intracellular trafficking induced by octreotide and pasireotide.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

Host Regulation of Ebola Virus Egress and Spread: Role of Cytoskeletal Filamin Proteins

Ebola virus (EBOV) causes severe hemorrhagic fever associated with high mortality rates in humans. EBOV and other filoviruses are emerging BSL‐4 pathogens for which the development of novel and effective therapeutics is urgently needed. Egress and spread of EBOV is mediated by the VP40 matrix protein (eVP40), whereby expression of eVP40 alone directs the production and egress of virus‐like particles (VLPs) that accurately mimics the budding process of live infectious virus. We have shown that eVP40 hijacks or recruits select host proteins to facilitate the budding process. On the other hand, we have also identified several host proteins that interact with eVP40 to negatively regulated eVP40‐mediated budding. Thus, a better understanding of the eVP40‐host interactome will provide important insights into the mechanisms of virus egress and spread, as well as inform about potential targets for the development of novel antiviral therapeutics. Here, we investigated the possible role for host filamin A and B proteins (actin crosslinking proteins that function in cell morphology and migration at the plasma membrane) in regulating both entry and egress of EBOV. Toward this end, we employed filamin A (FLNaKD) and filamin B knockdown cells (FLNbKD) to assess EBOV entry by using live EBOV, as well as recombinant viruses and/or pseudotypes expressing the EBOV surface glycoprotein (GP). We also used the KD cells to assess virus egress by using our well‐established eVP40 VLP budding assay. In sum, our results indicate that expression of host filamin proteins appears to be important for EBOV to efficiently complete different stages of its lifecycle.

Role of the Dimerization Domain of Filamin in Dictyostelium discoideum Response to Shear Flow

Molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation, which is critical in homeostasis and many diseases, are not well understood. Dictyostelium discoideum cells exposed to a brief burst of shear flow show rapid and transient activation of multiple components of the signal transduction network that participates in directed migration of these cells. Previous data from our laboratory demonstrated that actin crosslinking protein filamin is involved in the ability of cells to respond to shear flow. We also found that the actin‐binding domain is required for filamin’s function in this context. To determine if the dimerization domain (DD) is also required for filamin’s role in sensing/transmitting mechanical stimuli we generated a truncation construct of filamin lacking DD (FLNΔDD) and expressed it in wild‐type or filamin‐null cells. We found that FLNΔDDwas able to relocalize to the cortex of both wild‐type and filamin‐null cells following 2‐sec stimulation with shear flow, suggesting that dimerization between filamin molecules is not required for their recruitment to the cortex. To detect activation of the signal transduction network in the presence or absence of FLNΔDD, we used fluorescently‐tagged Ras binding domain biosensor that detects active Ras and was previously shown to relocalize to the cortex following mechanical stimulation. Surprisingly, FLNΔDDwas able to rescue the reduced response of filamin‐null cells to shear flow stimulation, suggesting that dimerization of filamin may not be needed for its ability to sense or transmit mechanical cues. However, the presence of FLNΔDD may also alter the overall organization of the cytoskeletal network, potentially explaining changes in mechanotransduction. We are currently investigating these possibilities.

Hematopoietic protein RELT is upregulated in human breast and lung cancers and binds the cytoskeletal protein Filamin A.

Abstract Receptor Expressed in Lymphoid Tissues (RELT), is a human TNFR superfamily member (TNFRSF19L) expressed most prominently in the hematopoietic system. Previous reports using RELT knockout mice indicate that RELT functions in part by inhibiting the proliferation and activation of T lymphocytes. RELT is an orphan receptor that has two homologous binding partners, RELL1 and RELL2, and collectively, these three proteins are referred to as RELT family members. This study sought to identify novel protein interactions with RELT family members and to compare the expression of RELT in normal and diseased human tissues. A yeast two-hybrid screen utilizing RELL1 as bait identified the cytoskeletal protein Filamin A (FLNA) as a prospective binding partner and physical interaction between FLNA and RELT family members was confirmed by in vitro co-immunoprecipitation. A truncated mutant of FLNA disrupts the ability of RELT family members to activate the p38 pathway and blocks the ability of RELT to induce death in human epithelial cells. Western blotting revealed endogenous RELT family members are most significantly upregulated in the breast cancer cell lines MDA-MB-231 and MCF-7, as well as the lung cancer cell line H358. Preliminary immunohistochemistry results reveal a higher intensity of RELT staining in both breast cancer and lung cancer human biopsies versus non-malignant tissue. Additionally, overexpression of RELT family members enhanced apoptosis in MDA-MB-231 and H358 cells as measured by X-gal morphology and luciferase assays. Collectively, these results further our understanding of signal transduction pathways associated with RELT family members and report the novel finding that RELT is upregulated in human breast and lung cancers. Supported by California Northstate University, College of Medicine, Seed Grant.

Molecular mechanisms involved in somatostatin receptor regulation in corticotroph tumors: the role of cytoskeleton and USP8 mutations.

Adrenocorticotropic hormone (ACTH)-secreting pituitary tumors mainly express somatostatin receptor 5 (SSTR5) since SSTR2 is downregulated by the elevated levels of glucocorticoids that characterize patients with Cushing's disease (CD). SSTR5 is the molecular target of pasireotide, the only approved pituitary tumor-targeted drug for the treatment of CD. However, the molecular mechanisms that regulate SSTR5 are still poorly investigated. This review summarizes the experimental evidence supporting the role of the cytoskeleton actin-binding protein filamin A (FLNA) in the regulation of SSTR5 expression and signal transduction in corticotroph tumors. Moreover, the correlations between the presence of somatic USP8 mutations and the expression of SSTR5 will be reviewed. An involvement of glucocorticoid-mediated β-arrestins modulation in regulating SSTRs expression and function in ACTH-secreting tumors will also be discussed.

Deletion of cardiac filamin C induces subcellular remodeling that inhibits mechanical force transmission

Dilated cardiomyopathy (DCM) is a common and deadly form of heart disease that is typically characterized by progressive thinning of ventricular walls, chamber dilation, and systolic dysfunction. DCM is often associated with mutations in genes encoding sarcomere or cytoskeleton proteins that confer contractile dysfunction and adverse cellular remodeling. The cytoskeletal protein Filamin C (FLNC) interacts with multiple proteins in the Z-disc and the costamere, suggesting that it contributes to costamere-based mechanotransduction in the heart.

Filamin A forms a complex with EphA2 and regulates EphA2 serine 897 phosphorylation and glioblastoma cell proliferation

EphA2 is phosphorylated on serine 897 (S897) in response to growth factors such as epidermal growth factor (EGF) and on tyrosine 588 (Y588) in response to its ligand ephrinA1, causing different cellular responses. In this study, we show that the actin-binding protein Filamin A forms a complex with EphA2 and promotes its S897 phosphorylation and glioblastoma cell proliferation. Suppression of Filamin A expression by siRNAs inhibited glioblastoma cell proliferation induced by EGF stimulation or overexpression of EphA2. Knockdown of Filamin A inhibited EGF-induced S897 phosphorylation of EphA2, whereas it had little effect on ephrinA1-induced Y588 phosphorylation of EphA2. Furthermore, Filamin A expression affected the subcellular localization of EphA2. This study suggests that Filamin A selectively promotes EphA2 S897 phosphorylation and plays an important role in glioblastoma cell proliferation.

Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Filamin A increases aggressiveness of human neuroblastoma.

BackgroundThe actin-binding protein filamin A (FLNA) regulates oncogenic signal transduction important for tumor growth, but the role of FLNA in the progression of neuroblastoma (NB) has not been explored.MethodsWe analyzed FLNA mRNA expression in the R2 NB-database and FLNA protein expression in human NB tumors. We then silenced FLNA expression in human SKNBE2 and IMR32 NB cells by lentiviral vector encoding shRNA FLNA and assayed the cells for proliferation, migration, colony, spheroid formation, and apoptosis. SKNBE2 xenografts expressing or lacking FLNA in BALB/c nude mice were analyzed by both routine histopathology and immunohistochemistry.ResultsWe observed shorter patient survival with higher expression of FLNA mRNA than patients with lower FLNA mRNA expression, and high-risk NB tumors expressed higher FLNA levels. Overexpression of FLNA increased proliferation of SH-SY5 NB cells. NB cell lines transfected with siRNA FLNA proliferated and migrated less, expressed lower levels of phosphorylated AKT and ERK1/2, formed smaller colonies and spheroids, as well as increased apoptosis. After inoculation of SKNBE2 cells infected with lentivirus expressing shRNA FLNA, size of NB tumors and number of proliferating cells were decreased. Furthermore, we identified STAT3 as an interacting partner of FLNA. Silencing FLNA mRNA reduced levels of NF-κB, STAT3 and MYCN, and increased levels of p53 and cleaved caspase 3.ConclusionInhibition of FLNA impaired NB cell signaling and function and reduced NB tumor size in vivo, suggesting that drugs targeting either FLNA or its interaction with STAT3 may be useful in the treatment of NB.

Developmental differences of in vitro cultured murine bone marrow- and fetal liver-derived megakaryocytes

Multiple lines of evidence support differences in the megakaryopoiesis during development. Murine in vitro models to study megakaryopoiesis employ cultured megakaryocytes MKs derived from adult bone marrow (BM) or fetal livers (FL) of mouse embryos. Mouse models allow to study the molecular basis for cellular changes utilizing conditional or knock-out models and permit further in vitro genetic or pharmacological manipulations. Despite being extensively used, MKs cultured from these two sources have not been systematically compared. In the present study, we compared BM- and FL-derived MKs, assessing their size, proplatelet production capacity, expression of common MK markers (αIIb, β3, GPIb α, β) and cytoskeletal proteins (filamin A, β1-tubulin, actin), the subcellular appearance of α-granules (VWF), membranes (GPIbβ) and cytoskeleton (F-actin) throughout in vitro development. We demonstrate that FL MKs although smaller in size, spontaneously produce more proplatelets than BM MKs and at earlier stages express more β1-tubulin. In addition, early FL MKs show increased internal GPIbβ staining and present higher GPIbβ (early and late) and VWF (late stages) total fluorescence intensity (TFI)/cell size than BM MKs. BM MKs have up-regulated TPO signaling corresponding to their bigger size and ploidy, without changes in c-Mpl. Expressing endogenous β1-tubulin or the presence of heparin improves BM MKs ability to produce proplatelets. These data suggest that FL MKs undergo cytoplasmic maturation earlier than BM MKs and that this, in addition to higher β1-tubulin levels and GPIb, supported with an extensive F-actin network, could contribute to more efficient proplatelet formation in vitro.

The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells.

Simple SummaryThe renal sodium chloride cotransporter (NCC) plays an important role in the total body electrolyte balance and blood pressure control. The regulation of this protein by the actin cytoskeleton has not been thoroughly studied. Here, we investigate a novel association between the actin cytoskeleton protein filamin A and the NCC using a mouse cellular model and in the native kidney. Our results show for the first time that the disruption of the actin cytoskeleton reduces NCC activity and filamin A plays an essential role in NCC protein expression in cells of the distal convoluted tubule. We further show that the pharmacological inhibition of the Ca2+/calmodulin dependent protein kinase II (CAMKII) augments NCC protein expression. These results introduce a new mechanism for the regulation of the NCC.The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

Isoform-specific functions of synaptopodin-2 variants in cytoskeleton stabilization and autophagy regulation in muscle under mechanical stress

Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.

Bipolar disorder with Melnick\u2013Needles syndrome and periventricular nodular heterotopia: two case reports\xa0and\xa0a review of the\xa0literature

BackgroundMelnick–Needles syndrome and periventricular nodular heterotopia are two usually mutually exclusive phenotypes of F-actin-binding cytoskeletal phosphoprotein Filamin-A mutations. Melnick–Needles syndrome is a rare X-linked condition that is lethal in males and shows great phenotypic variability in affected females. It is caused by mutations in Filamin-A gene, which encodes the protein Filamin A. Defects of the human Filamin-A gene also cause X-linked periventricular nodular heterotopia, a malformation of neuronal migration characterized by nodules of neurons in inappropriate location adjacent to the walls of the lateral ventricles.Case presentationWe report on two Caucasian adolescent females, sisters, diagnosed with Melnick–Needles syndrome and bilateral periventricular nodular heterotopia, who developed bipolar disorder and somatic symptoms disorder at a young age. We also present a review of the literature about mental disorders associated with periventricular nodular heterotopia. Our report shows that patients presenting with atypical and heterogeneous psychiatric disease may have an underrecognized anatomical brain abnormality on genetic basis.ConclusionsWe found records of psychiatric disorders associated with periventricular nodular heterotopia; nevertheless, this is the first report of bipolar disorder occurring in individuals with periventricular nodular heterotopia, and the first report of any psychiatric disorder in individuals affected by Melnick–Needles syndrome. In conclusion, this case report may contribute to characterizing the phenotype of this very rare syndrome.

A CRISPR/Cas9 strategy for the generation of a FLNC knockout hESC line (WAe009-A-70) to model dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy

The FLNC gene encodes the sarcomeric protein filamin C which plays a central role in cardiomyocytes. Truncating FLNC mutations (stop or frameshift etc.) also cause dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To further understand the exact role of FLNC in DCM, we have generated a human FLNC knockout cell line from the original embryonic stem cell line H9 by CRISPR/Cas9 gene editing technology in this study. The establishment cell line WAe009-A-70 have a compound heterozygous 4 bp deletion/13 bp deletion in the exon 1 of FLNC which resulted in a frameshift in the translation of FLNC. No filamin C protein was detected in cardiomyocytes differentiated from this cell line. Moreover, WAe009-A-70 also expressed pluripotency markers, maintained the ability to differentiate into the three germ layers in vitro and had a normal karyotype.

Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation

BackgroundCytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of β subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated. MethodsBinding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking. ResultsThe αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions. ConclusionOur findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state. General significanceFilamin binding to both α and β CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.

Differential Roles of Actin Crosslinking Proteins Filamin and α-Actinin in Shear Flow-Induced Migration of Dictyostelium discoideum.

Shear flow-induced migration is an important physiological phenomenon experienced by multiple cell types, including leukocytes and cancer cells. However, molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation are not well understood. Dictyostelium discoideum social amoeba, a well-established model for studying amoeboid-type migration, also exhibits directional motility when exposed to shear flow, and this behavior is preceded by rapid and transient activation of the same signal transduction network that is activated by chemoattractants. The initial response, which can also be observed following brief 2 s stimulation with shear flow, requires an intact actin cytoskeleton; however, what aspect of the cytoskeletal network is responsible for sensing and/or transmitting the signal is unclear. We investigated the role of actin crosslinkers filamin and α-actinin by analyzing initial shear flow-stimulated responses in cells with or without these proteins. Both filamin and α-actinin showed rapid and transient relocalization from the cytosol to the cortex following shear flow stimulation. Using spatiotemporal analysis of Ras GTPase activation as a readout of signal transduction network activity, we demonstrated that lack of α-actinin did not reduce, and, in fact, slightly improved the response to acute mechanical stimulation compared to cells expressing α-actinin. In contrast, shear flow-induced Ras activation was significantly more robust in filamin-null cells rescued with filamin compared to cells expressing empty vector. Reduced responsiveness appeared to be specific to mechanical stimuli and was not due to a change in the basal activity since response to global stimulation with a chemoattractant and random migration was comparable between cells with or without filamin. Finally, while filamin-null cells rescued with filamin efficiently migrated upstream when presented with continuous flow, cells lacking filamin were defective in directional migration. Overall, our study suggests that filamin, but not α-actinin, is involved in sensing and/or transmitting mechanical stimuli that drive directed migration; however, other components of the actin cytoskeleton likely also contribute to the initial response since filamin-null cells were still able to activate the signal transduction network. These findings could have implications for our fundamental understanding of shear flow-induced migration of leukocytes, cancer cells and other amoeboid-type cells.

Periostin/Filamin-A: A Candidate Central Regulatory Axis for Valve Fibrogenesis and Matrix Compaction.

BackgroundDiscoveries in the identification of transcription factors, growth factors and extracellular signaling molecules have led to the detection of downstream targets that modulate valvular tissue organization that occurs during development, aging, or disease. Among these, matricellular protein, periostin, and cytoskeletal protein filamin A are highly expressed in developing heart valves. The phenotype of periostin null indicates that periostin promotes migration, survival, and differentiation of valve interstitial cushion cells into fibroblastic lineages necessary for postnatal valve remodeling/maturation. Genetically inhibiting filamin A expression in valve interstitial cushion cells mirrored the phenotype of periostin nulls, suggesting a molecular interaction between these two proteins resulted in poorly remodeled valve leaflets that might be prone to myxomatous over time. We examined whether filamin A has a cross-talk with periostin/signaling that promotes remodeling of postnatal heart valves into mature leaflets.ResultsWe have previously shown that periostin/integrin-β1 regulates Pak1 activation; here, we revealed that the strong interaction between Pak1 and filamin A proteins was only observed after stimulation of VICs with periostin; suggesting that periostin/integrin-β-mediated interaction between FLNA and Pak1 may have a functional role in vivo. We found that FLNA phosphorylation (S2152) is activated by Pak1, and this interaction was observed after stimulation with periostin/integrin-β1/Cdc42/Rac1 signaling; consequently, FLNA binding to Pak1 stimulates its kinase activity. Patients with floppy and/or prolapsed mitral valves, when genetically screened, were found to have point mutations in the filamin A gene at P637Q and G288R. Expression of either of these filamin A mutants failed to increase the magnitude of filamin A (S2152) expression, Pak1-kinase activity, actin polymerization, and differentiation of VICs into mature mitral valve leaflets in response to periostin signaling.ConclusionPN-stimulated bidirectional interaction between activated FLNA and Pak1 is essential for actin cytoskeletal reorganization and the differentiation of immature VICs into mature valve leaflets.

The Role of Zyxin in Regulating Platelet Cytoskeleton Distribution

To investigate the regulatory effect of zyxin on the distribution of platelet cytoskeleton. Platelets were isolated from zyxin-knockout (Zyx-/-) and wild type (WT) mice respectively and corresponding platelet cytoskeleton components were separated. The expressions of β-actin, α-actinin, filamin A and myosin ⅡA in cytoskeleton components were detected by Western blot. Actin polymerization was induced by the actin polymerization inducer Jasplakinolide (Jas) in WT and Zyx-/- platelets. Also, the expressions of the cytoskeleton proteins in cytoskeleton components were detected by Western blot. WT and Zyx-/- platelets were allowed to spread on fibrinogen-coated surface. Platelet F-actin was labeled with Alexa Fluor 488-conjugated phalloidin and the fluorescent intensity was compared between WT and Zyx-/- platelets. After zyxin gene was knockout, the expressions of cytoskeleton proteins β-actin, α-actinin, filamin A, and myosin Ⅱ A in resting and Jas-induced platelets were significantly increased. In the platelet spreading on fibrinogen surface, F-actin was increased in Zyx-/- platelets as compared with that in WT platelets. Zyxin significantly regulates the distribution of platelet cytoskeleton, which plays an important role in maintaining platelet cytoskeleton homeostasis.