Jun Qin

Crucial to homeostasis, vascular barrier function depends upon coordinated interplay between endothelial (ECs) and mural cells, including vascular smooth muscle cells (vSMCs) and pericytes, that stabilize the vasculature. Kindlin‐2 (K2) is an integrin co‐activator regulating various cellular responses, but its role in mural cell‐dependent vascular stabilization is unknown. The role of K2 in mural cell‐mediated regulation of vascular barrier function was investigated. Vascular permeability was assessed in SMC/pericyte‐specific tamoxifen‐inducible K2 knockout ( Fermt2 ^Δ/Δ Myh11‐CreERT2 ⁺ ROSA26‐floxed STOP eYFP ^+/+ ), lineage tracing mice (K2 ^Δ/Δ ) and their K2 wild‐type littermates (K2 ^WT/WT ). In Miles assays, K2 ^Δ/Δ mice showed increased basal by (20%–75%) and vEGF, PAF, or mustard oil‐induced vascular permeability of Evans blue into the skin (by 2‐ to 3‐fold) compared to K2 ^WT/WT mice. In LPS‐induced sepsis, vascular leakage into the lungs and liver was 50% higher in K2 ^Δ/Δ mice than in K2 ^WT/WT littermates. The enhanced vascular leakiness in K2 ^Δ/Δ mice was due to aberrant vasculature characterized by decreased coverage with vSMCs. In ex vivo experiments, K2 ^Δ/Δ aortic vSMCs and brain pericytes had severely reduced β1 and β3 integrin activation, leading to attenuated adhesion to integrin ligands (by ~60%–80%) compared to the K2 ^WT/WT cells. K2 ^Δ/Δ vSMCs showed diminished interactions with ECs during endothelial tube formation, dedifferentiation, and enhanced apoptosis. The same effect was observed in human aortic vSMCs upon 50% K2 downregulation with K2‐specific shRNA. In contrast to K2 ^WT/WT , the K2 ^Δ/Δ pericytes failed to support barrier formation by brain microvascular ECs in the ex vivo blood–brain barrier model. Together, K2 as an integrin coactivator sustains mural cell functions, contributing to vascular stabilization.

BACKGROUND: FLNC (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. METHODS: We 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. RESULTS: Our 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. CONCLUSIONS: Our 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.

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.

Zero-index metamaterials (ZIMs) feature a uniform electromagnetic mode over a large area in arbitrary shapes, enabling many applications including high-transmission supercouplers with arbitrary shapes, direction-independent phase matching for nonlinear optics, and collective emission of many quantum emitters. However, most ZIMs reported till date are passive, with no method for the dynamic modulation of their electromagnetic properties. Here, we design and fabricate a magnetically tunable ZIM consisting of yttrium iron garnet (YIG) pillars sandwiched between two copper clad laminates in the microwave regime. By harnessing the Cotton-Mouton effect of YIG, the metamaterial was successfully toggled between gapless and bandgap states, leading to a "phase transition" between a zero-index phase and a single negative phase of the metamaterial. Using an S-shaped ZIM supercoupler, we experimentally demonstrated a tunable supercoupling state with a low intrinsic loss of 0.95 dB and a high extinction ratio of up to 30.63 dB at 9 GHz. Our work enables dynamic modulation of the electromagnetic characteristics of ZIMs, enabling various applications in tunable linear, nonlinear, quantum and nonreciprocal electromagnetic devices.

Talin-induced integrin binding to extracellular matrix ligands (integrin activation) is the key step to trigger many fundamental cellular processes including cell adhesion, cell migration, and spreading. Talin is widely known to use its N-terminal head domain (talin-H) to bind and activate integrin, but how talin-H operates in the context of full-length talin and its surrounding remains unknown. Here we show that while being capable of inducing integrin activation, talin-H alone exhibits unexpectedly low potency versus a constitutively activated full-length talin. We find that the large C-terminal rod domain of talin (talin-R), which otherwise masks the integrin binding site on talin-H in inactive talin, dramatically enhances the talin-H potency by dimerizing activated talin and bridging it to the integrin co-activator kindlin-2 via the adaptor protein paxillin. These data provide crucial insight into the mechanism of talin and its cooperation with kindlin to promote potent integrin activation, cell adhesion, and signaling.

Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.

Ras suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating fundamental cell adhesion processes and tumor development. Rsu-1 interacts with a zinc-finger type multi LIM domain-containing adaptor protein PINCH-1 involved in the integrin-mediated consensus adhesome but not with highly homologous isoform PINCH-2. However, the structural basis for such specific interaction and regulatory mechanism remains unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture with eight LRRs shielded by the N- and C-terminal capping modules. We show that a large conserved concave surface of the Rsu-1 LRR domain recognizes the PINCH-1 LIM5 domain, and that the C-terminal non-LIM region of PINCH-2 but not PINCH-1 sterically disfavors the Rsu-1 binding. We further show that Rsu-1 can be assembled, via PINCH-1-binding, into a tight hetero-pentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2 that constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Consistently, our mutagenesis and cell biological data consolidate the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading. Our results provide a crucial molecular insight into Rsu-1-mediated cell adhesion with implication on how it may regulate tumorigenic growth.