Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function

BackgroundFLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood.MethodsWe conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations.ResultsOur findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components.ConclusionsOur data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions

Evidence for Multisite Ligand Binding and Stretching of Filamin by Integrin and Migfilin

Filamin, a large cytoskeletal adaptor, connects plasma membrane to cytoskeleton by binding to transmembrane receptor integrin and actin. Seven of 24 filamin immunoglobulin repeats have conserved integrin binding sites, of which repeats 19 and 21 were shown to be autoinhibited by their adjacent repeats 18 and 20, respectively. Here we show using nuclear magnetic resonance spectroscopy that the autoinhibition can be relieved by integrin or integrin regulator migfilin. We further demonstrate that repeats 19 and 21 can simultaneously engage ligands. The data suggest that filamin is mechanically stretched by integrin or migfilin via a multisite binding mechanism for regulating cytoskeleton and integrin-mediated cell adhesion

Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2)

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not β3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2µ (clathrin AP2µ) and β-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2µ- and PAR2-β-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis.</jats:p

Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2)

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not &beta;3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2&micro; (clathrin AP2&micro;) and &beta;-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2&micro;- and PAR2-&beta;-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis

Mechanisms of talin-dependent integrin signaling and crosstalk

AbstractCells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins. Extracellular or intracellular signals emanating from different signaling cascades cause inside-out signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton. Various integrin subfamilies communicate with each other and growth factor receptors under diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an essential mediator of integrin activation, much of the integrin crosstalk would therefore be influenced by talin. However, despite the existence of an extensive body of knowledge on the role of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in regulating inter-integrin communication is limited. This review will focus on the structure of talin, its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

Structural mechanism of integrin inactivation by filamin

The activation of heterodimeric (α/β) integrin is crucial for regulating cell adhesion. Binding of talin to the cytoplasmic face of integrin activates the receptor, but how integrin is properly maintained in resting state to counterbalance its activation for regulating adhesion dynamics remains obscure. We report the structure of cytoplasmic domain of human integrin αIIbβ3 bound to its inhibitor, the immunoglobin repeat 21 of filamin A (FLNa-Ig21). The structure reveals an unexpected ternary complex where FLNa-Ig21 not only binds to previously predicted C-terminus of integrin β3 cytoplasmic tail (CT) but also engages N-terminal helices of αIIb and β3 CTs to stabilize an inter-CT clasp that helps restrain the integrin in a resting state. Combined with functional data, the structure reveals a novel mechanism of filamin-mediated retention of inactive integrin, suggesting a new framework for understanding regulation of integrin activation and adhesion. Keywords Integrin; filamin; cell adhesion; NMR Integrins are a family of major cell surface receptors that mediate essentially every lif

A mechanism of platelet integrin aIIbb3 outside-in signaling through a novel integrin aIIb subunit-filamin-actin linkage

The communication of talin-activated integrin aIIbb3 with cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin cross-linker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive aIIbb3, is displaced from aIIbb3 by talin to promote the integrin activation (inside-out signaling) and how filamin further functions remains unresolved. Here we show that while associating with the inactive aIIbb3, filamin also associates with the talin-bound active aIIbb3 to mediate platelet spreading. FRET-based analysis reveals that while associating with both aIIb and b3 cytoplasmic tails (CTs) to maintain the inactive aIIbb3, filamin is spatiotemporally re-arranged to associate with aIIb CT alone on activated aIIbb3. Consistently, confocal cell imaging indicates that integrin a CT-linked filamin gradually delocalizes from b CT-linked focal adhesion marker - vinculin likely due to the separation of integrin a/b CTs occurring during integrin activation. High-resolution crystal and NMR structure determinations unravel that the activated integrin aIIb CT binds to filamin via a striking a-helix→b-strand transition with strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin aIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of aIIbb3, phosphorylation of FAK/Src kinases, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.</jats:p