Mechanically operated signalling scaffolds.

Cellular signalling is a complex process and involves cascades of enzymes that, in response to a specific signal, give rise to exact cellular responses. Signalling scaffold proteins organise components of these signalling pathways in space and time to co-ordinate signalling outputs. In this review we introduce a new class of mechanically operated signalling scaffolds that are built into the cytoskeletal architecture of the cell. These proteins contain force-dependent binary switch domains that integrate chemical and mechanical signals to introduce quantised positional changes to ligands and persistent alterations in cytoskeletal architecture providing mechanomemory capabilities. We focus on the concept of spatial organisation, and how the cell organises signalling molecules at the plasma membrane in response to specific signals to create order and distinct signalling outputs. The dynamic positioning of molecules using binary switches adds an additional layer of complexity to the idea of scaffolding. The switches can spatiotemporally organise enzymes and substrates dynamically, with the introduction of ∼50 nm quantised steps in distance between them as the switch patterns change. Together these different types of signalling scaffolds and the proteins engaging them, provide a way for an ordering of molecules that extends beyond current views of the cell.

Caskin2 is a novel talin and Abi1-binding protein that promotes cell motility.

Talin couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE Regulatory Complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in CMSCs. Taken together, our results identify Caskin2 as a novel talin-binding protein that may not only connect integrin-mediated adhesion to actin polymerization, but could also play a role in crosstalk between integrins and microtubules.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein.

Protein kinase A (PKA) is a ubiquitous, promiscuous kinase whose activity is specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), suggesting the existence of one or more FA AKAPs. Using a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1 to R13. Direct binding assays and NMR spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Experiments with single molecules and in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. Finally, talin mutations that disrupt PKA binding also decrease levels of total and phosphorylated PKA RII subunits as well as phosphorylation of VASP, a known PKA substrate, within FA. These observations identify a mechanically gated anchoring protein for PKA, a force-dependent binding partner for talin1, and a potential pathway for adhesion-associated mechanotransduction.

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5ß1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling.

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

Role of the Ste20-like kinase SLK in podocyte adhesion.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

ATG8 inhibited endometriosis formation by regulating Treg cells differentiation via integrin α4ß1 and Talin-1 interaction.

RESEARCH QUESTION: What is the relationship between ATG8 and integrin α4ß1, Talin-1, and Treg cell differentiation, and the effects on endometriosis (EMS)? DESIGN: First, the correlation between the ATG8, Talin-1, integrin α4ß1, and differentiation of Treg cells and EMS was examined in clinical samples. Human peripheral blood mononuclear cells (PBMC) and endometrial stromal cells were extracted and identified, oe-ATG8 and oe-integrin α4ß1 were transfected to overexpress ATG8 and integrin α4ß1, and Tregs cell differentiation and endometrial stromal cells (ESC) function were detected. In addition, the molecular mechanism by which ATG8 inhibited EMS disease progression at the molecular and animal levels was investigated. RESULTS: ATG8 expression was negatively correlated with positive proportion of Tregs cells (P = 0.0463). The expression of Talin-1 and integrin-α4ß1 (both P < 0.0001) in PBMC decreased significantly after oe-ATG8 transfection, whereas the Treg cells' positive rate significantly increased (P = 0.0003). The ESC proliferation, adhesion, migration, and invasion (all P < 0.0001) declined after co-culture with Treg cells that underwent oe-ATG8 transfection. The expression of Talin-1 (P = 0.0025) and integrin-α4ß1 (P = 0.0002) in PBMC increased significantly after oe-integrin α4ß1 and oe-ATG8 transfection. In addition, this transfection reversed the corresponding regulation of oe-ATG8 transfection. Finally, animal experiments in vivo confirmed that ATG8 inhibited EMS disease progression. CONCLUSION: The ATG8 regulated Treg cell differentiation and inhibited EMS formation by influencing the interaction between integrin α4ß1 and Talin-1.

Talin mechanotransduction in disease.

Talin protein (Talin 1/2) is a mechanosensitive cytoskeleton protein. The unique structure of the Talin plays a vital role in transmitting mechanical forces. Talin proteins connect the extracellular matrix to the cytoskeleton by linking to integrins and actin, thereby mediating the conversion of mechanical signals into biochemical signals and influencing disease progression as potential diagnostic indicators, therapeutic targets, and prognostic indicators of various diseases. Most studies in recent years have confirmed that mechanical forces also have a crucial role in the development of disease, and Talin has been found to play a role in several diseases. Still, more studies need to be done on how Talin is involved in mechanical signaling in disease. This review focuses on the mechanical signaling of Talin in disease, aiming to summarize the mechanisms by which Talin plays a role in disease and to provide references for further studies.

The Association Between Higher Expression of Talin-1 and the Reduced Survival Rate in Ovarian Serous Carcinoma Patients.

Background & Objective: Talin-1 is a constituent of the multiprotein adhesion complexes that play main role in the formation of tumors and migration in different types of malignancies. The present study aimed to assess expression and prognostic significance of the talin-1 protein in ovarian serous carcinoma (OSC) patients. Methods: The expression of talin-1 in mRNA and its protein levels were investigated for ovarian cancer (OC) by using bioinformatics tools, including Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Gene Expression Database of Normal and Tumor Tissue 2 (GENT2), and The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) databases. Thereafter, immunohistochemical (IHC) staining was used to study the expression patterns of the talin-1 protein using 46 paraffin-embedded OSC tissue specimens, 25 benign tumors, and 20 normal tissues, which were assembled in tissue microarrays (TMAs). We also assessed the potential association between the expression of the talin-1 protein, various clinicopathological parameters, and survival outcomes. Results: Our IHC examination for talin-1 was significantly overexpressed in OSC tissues compared to benign tumors and normal tissues. The Kaplan-Meier survival analysis has also indicated statistically significant differences in terms of disease-specific survival (DSS) and progression-free survival (PFS) between the patients with high and low expression levels of talin-1, respectively. Conclusion: The talin-1 protein was overexpressed in OSC tissues, and a high expression level of talin-1 was found to be significantly associated with tumor aggressiveness and poorer DSS or PFS. Therefore, talin-1 may serve as a molecular marker of cancer progression and a novel prognostic biomarker in these patients.

Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub.

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional 'tug-of-war' on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

KANK1 shapes focal adhesions by orchestrating protein binding, mechanical force sensing, and phase separation.

Focal adhesions (FAs) are dynamic protein assemblies that connect cytoskeletons to the extracellular matrix and are crucial for cell adhesion and migration. KANKs are scaffold proteins that encircle FAs and act as key regulators of FA dynamics, but the molecular mechanism underlying their specified localization and functions remains poorly understood. Here, we determine the KANK1 structures in complex with talin and liprin-ß, respectively. These structures, combined with our biochemical and cellular analyses, demonstrate how KANK1 scaffolds the FA core and associated proteins to modulate the FA shape in response to mechanical force. Additionally, we find that KANK1 undergoes liquid-liquid phase separation (LLPS), which is important for its localization at the FA edge and cytoskeleton connections to FAs. Our findings not only indicate the molecular basis of KANKs in bridging the core and periphery of FAs but also provide insights into the LLPS-mediated dynamic regulation of FA morphology.

[Molecular dynamics simulation of force-regulated interaction between talin and Rap1b].

The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the ß4 sheet of the F0 domain was pulled away from the original ß1-ß4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP 54-ARG 41 and GLN 18-THR 65, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.

CdGAP is a talin-binding protein and a target of TGF-ß signaling that promotes HER2-positive breast cancer growth and metastasis.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastasis, which is the leading cause of death in breast cancer patients. Here, we show that Cdc42 GTPase-activating protein (CdGAP) promotes tumor formation and metastasis to lungs in the HER2-positive (HER2+) murine breast cancer model. CdGAP facilitates intravasation, extravasation, and growth at metastatic sites. CdGAP depletion in HER2+ murine primary tumors mediates crosstalk with a Dlc1-RhoA pathway and is associated with a transforming growth factor ß (TGF-ß)-induced EMT transcriptional signature. CdGAP is positively regulated by TGF-ß signaling during EMT and interacts with the adaptor talin to modulate focal adhesion dynamics and integrin activation. Moreover, HER2+ breast cancer patients with high CdGAP mRNA expression combined with a high TGF-ß-EMT signature are more likely to present lymph node invasion. Our results suggest CdGAP as a candidate therapeutic target for HER2+ metastatic breast cancer by inhibiting TGF-ß and integrin/talin signaling pathways.

Talin1 regulates glucose metabolism and endometrial receptivity via GLUT-4 in patients with polycystic ovary syndrome and insulin resistance.

Objectives: Talin1 is a cytoskeletal protein and is localized between cells and the extracellular matrix. This study aimed to investigate the mechanism by which Talin1 affects glucose metabolism and endometrial receptivity via glucose transporter proteins-4 (GLUT-4) in patients with polycystic ovary syndrome (PCOS) and insulin resistance (IR). Methods: We examined the expression of Talin1 and GLUT4 in the receptive endometrium of PCOS-IR and control patients. GLUT4 expression was examined after silencing and overexpression of Talin1 in Ishikawa cells. We validated the interaction between Talin1 and GLUT-4 proteins using a co-immunoprecipitation (Co-IP) assay. After successfully establishing the C57BL/6j mouse model of PCOS-IR, the expression of Talin1 and GLUT-4 were examined in PCOS-IR and control mice. The effect of Talin1 on embryo implantation and the number of live births in mice were examined. Results: Our study found low expression of Talin1 and GLUT-4 in the receptive endometrium of PCOS-IR patients compared to that in control patients (p < 0.01). The level of GLUT-4 expression decreased after silencing Talin1 in Ishikawa cells and increased after overexpression of Talin1. Co-IP results showed that Talin1 interacts with GLUT-4 protein. We successfully established a PCOS-IR C57BL/6j mouse model and found that Talin1 and GLUT-4 expression in the receptive endometrium of PCOS-IR mice were lower than that in control mice (p < 0.05). In vivo experiments confirmed that the knockdown of Talin1 affects embryo implantation (p < 0.05) and live birth rate in mice (p < 0.01). Conclusions: Talin1 and GLUT-4 expression were decreased in the endometrium of PCOS-IR patients, and Talin1 may affect glucose metabolism and endometrial receptivity through GLUT4.

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.

Clutching at Guidance Cues: The Integrin-FAK Axis Steers Axon Outgrowth.

Integrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin-FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury.

Distinct bidirectional regulation of LFA1 and α4ß7 by Rap1 and integrin adaptors in T cells under shear flow.

Bidirectional control of integrin activation plays crucial roles in cell adhesive behaviors, but how integrins are specifically regulated by inside-out and outside-in signaling has not been fully understood. Here, we report distinct bidirectional regulation of major lymphocyte homing receptors LFA1 and α4ß7 in primary T cells. A small increase of Rap1 activation in L-selectin-mediated tether/rolling was boosted by the outside-in signaling from ICAM1-interacting LFA1 through subsecond, simultaneous activation of Rap1 GTPase and talin1, but not kindlin-3, resulting in increased capture and slowing. In contrast, none of them were required for tether/rolling by α4ß7 on MAdCAM1. High Rap1 activation with chemokines or the loss of Rap1-inactivating proteins Rasa3 and Sipa1 increased talin1/kindlin-3-dependent arrest with high-affinity binding of LFA1 to membrane-anchored ICAM1. However, despite increased affinity of α4ß7, activated Rap1 severely suppressed adhesion on MAdCAM1 under shear flow, indicating the critical importance of a sequential outside-in/inside-out signaling for α4ß7.

Inhibition of talin-induced integrin activation by a double-hit stapled peptide.

Integrins are ubiquitously expressed cell-adhesion proteins. Activation of integrins is triggered by talin through an inside-out signaling pathway, which can be driven by RAP1-interacting adaptor molecule (RIAM) through its interaction with talin at two distinct sites. A helical talin-binding segment (TBS) in RIAM interacts with both sites in talin, leading to integrin activation. The bispecificity inspires a "double-hit" strategy for inhibiting talin-induced integrin activation. We designed an experimental peptidomimetic inhibitor, S-TBS, derived from TBS and containing a molecular staple, which leads to stronger binding to talin and inhibition of talin:integrin interaction. The crystallographic study validates that S-TBS binds to the talin rod through the same interface as TBS. Moreover, the helical S-TBS exhibits excellent cell permeability and effectively suppresses integrin activation in cells in a talin-dependent manner. Our results shed light on a new class of integrin inhibitors and a novel approach to design multi-specific peptidomimetic inhibitors.

Assessment of the feed additive consisting of thaumatin for all animal species for the renewal of its authorisation (ADISSEO France S.A.S.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for the renewal of authorisation of thaumatin as a sensory additive (flavouring compound) for all animal species. The applicant requested a change in the authorising regulation for the minimum content of nitrogen and protein in the specification of the additive. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) confirms that the use of thaumatin under the current authorised conditions of use is safe for the target species, the consumers and the environment. This conclusion can be extended to the newly proposed specification. Due to its proteinaceous nature, the additive is considered to be a respiratory sensitiser. Thaumatin is not irritant to the eyes and the skin. In the absence of data, no conclusion on skin sensitisation could be made. The proposed modification of the specification of the additive is not considered to have an impact on the efficacy of thaumatin.

The structural basis of the talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions.

Adhesion between cells and the extracellular matrix is mediated by heterodimeric (αß) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organizes cytosolic signalling proteins into discrete complexes on ß-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we adapted a non-covalent crystallographic chaperone to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN region of KANK1 contains a novel motif where a ß-hairpin stabilizes the α-helical region, explaining both its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localizes throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.

TLN1 synergizes with ITGA5 to ameliorate cardiac microvascular endothelial cell dysfunction.

The complex process of atherosclerosis is thought to begin with endothelial cell dysfunction, and advanced atherosclerosis is the underlying cause of coronary artery disease (CAD). Uncovering the underlying mechanisms of CAD-related endothelial cell injury may contribute to the treatment. Cardiac microvascular endothelial cells (CMVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic an injury model. The involvement of Talin-1 (TLN1) and integrin alpha 5 (ITGA5) in the proliferation, apoptosis, angiogenesis, inflammatory response, and oxidative stress in CMVECs were assessed. TLN1 overexpression assisted CMVECs in resistance to ox-LDL stimulation, with alleviated cell proliferation and angiogenesis, reduced apoptosis, inflammatory response, and oxidative stress. TLN1 overexpression triggered increased ITGA5, and ITGA5 knockdown reversed the effects of TLN1 overexpression on the abovementioned aspects. Together, TLN1 synergized with ITGA5 to ameliorate the dysfunction in CMVECs. This finding suggests their probable involvement in CAD, and increasing their levels is beneficial to disease relief.