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1.
Proc Natl Acad Sci U S A ; 121(13): e2314947121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38513099

RESUMO

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.


Assuntos
Proteínas de Ancoragem à Quinase A , Adesões Focais , Adesões Focais/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Talina/metabolismo , Mecanotransdução Celular , Adesão Celular/fisiologia , Integrinas/metabolismo , Ligação Proteica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo
2.
Reprod Biomed Online ; 48(3): 103646, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38290387

RESUMO

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.


Assuntos
Endometriose , Integrina alfa4beta1 , Animais , Feminino , Humanos , Integrina alfa4beta1/metabolismo , Linfócitos T Reguladores , Talina/genética , Talina/metabolismo , Leucócitos Mononucleares/metabolismo , Diferenciação Celular , Progressão da Doença , Adesão Celular
3.
J Exp Clin Cancer Res ; 43(1): 27, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38254102

RESUMO

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.


Assuntos
Benzamidas , Carcinoma Epitelial do Ovário , Adesão Celular , Histona Desacetilase 1 , Histona Desacetilase 2 , Neoplasias Ovarianas , Neoplasias Peritoneais , Animais , Feminino , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Anticorpos Monoclonais , Carcinoma Epitelial do Ovário/metabolismo , Epitélio , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas , Histona Desacetilase 1/metabolismo , Integrina alfa5 , Integrina beta1/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Proteômica , Piridinas , Talina/genética , Talina/metabolismo , Histona Desacetilase 2/metabolismo , Adesão Celular/genética
4.
Physiol Rep ; 12(1): e15897, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38163671

RESUMO

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.


Assuntos
Nefrose , Podócitos , Camundongos , Animais , Podócitos/metabolismo , Paxilina/metabolismo , Vinculina/metabolismo , Talina/genética , Talina/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Doxorrubicina/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo
5.
Int J Biochem Cell Biol ; 166: 106490, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37914021

RESUMO

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.


Assuntos
Mecanotransdução Celular , Talina , Talina/química , Talina/metabolismo , Integrinas/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Adesão Celular/fisiologia
6.
Nat Struct Mol Biol ; 30(12): 1913-1924, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38087085

RESUMO

Integrin affinity regulation, also termed integrin activation, is essential for metazoan life. Although talin and kindlin binding to the ß-integrin cytoplasmic tail is indispensable for integrin activation, it is unknown how they achieve this function. By combining NMR, biochemistry and cell biology techniques, we found that talin and kindlin binding to the ß-tail can induce a conformational change that increases talin affinity and decreases kindlin affinity toward it. We also discovered that this asymmetric affinity regulation is accompanied by a direct interaction between talin and kindlin, which promotes simultaneous binding of talin and kindlin to ß-tails. Disrupting allosteric communication between the ß-tail-binding sites of talin and kindlin or their direct interaction in cells severely compromised integrin functions. These data show how talin and kindlin cooperate to generate a small but critical population of ternary talin-ß-integrin-kindlin complexes with high talin-integrin affinity and high dynamics.


Assuntos
Integrinas , Talina , Animais , Talina/química , Talina/metabolismo , Integrinas/metabolismo , Sítios de Ligação , Ligação Proteica
7.
Sci Rep ; 13(1): 22368, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38102166

RESUMO

The intestinal immune response is crucial in maintaining a healthy gut, but the enhanced migration of macrophages in response to pathogens is a major contributor to disease pathogenesis. Integrins are ubiquitously expressed cellular receptors that are highly involved in immune cell adhesion to endothelial cells while in the circulation and help facilitate extravasation into tissues. Here we show that specific deletion of the Tln1 gene encoding the protein talin-1, an integrin-activating scaffold protein, from cells of the myeloid lineage using the Lyz2-cre driver mouse reduces epithelial damage, attenuates colitis, downregulates the expression of macrophage markers, decreases the number of differentiated colonic mucosal macrophages, and diminishes the presence of CD68-positive cells in the colonic mucosa of mice infected with the enteric pathogen Citrobacter rodentium. Bone marrow-derived macrophages lacking expression of Tln1 did not exhibit a cell-autonomous phenotype; there was no impaired proinflammatory gene expression, nitric oxide production, phagocytic ability, or surface expression of CD11b, CD86, or major histocompatibility complex II in response to C. rodentium. Thus, we demonstrate that talin-1 plays a role in the manifestation of infectious colitis by increasing mucosal macrophages, with an effect that is independent of macrophage activation.


Assuntos
Colite , Infecções por Enterobacteriaceae , Animais , Camundongos , Citrobacter rodentium , Colite/genética , Colite/prevenção & controle , Colo/patologia , Células Endoteliais/metabolismo , Infecções por Enterobacteriaceae/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Talina/genética , Talina/metabolismo
8.
Nat Commun ; 14(1): 8468, 2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38123541

RESUMO

Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable ß-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study elucidates a force transmission mechanism, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.


Assuntos
Actinas , Talina , Actinas/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/metabolismo , Integrinas/metabolismo , Adesões Focais/metabolismo
9.
PLoS Genet ; 19(12): e1011089, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38150455

RESUMO

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Talina/metabolismo , Axônios/metabolismo , Vinculina , Regeneração Nervosa/genética , Fosforilação , Quinases Associadas a rho/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo
10.
J Am Chem Soc ; 145(45): 24459-24465, 2023 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-38104267

RESUMO

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.


Assuntos
Mecanotransdução Celular , Talina , Animais , Adesão Celular , Talina/metabolismo , Mecanotransdução Celular/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Mamíferos/metabolismo
11.
Cell Death Dis ; 14(10): 709, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37903776

RESUMO

Insufficient pancreatic ß-cell mass and reduced insulin expression are key events in the pathogenesis of diabetes mellitus (DM). Here we demonstrate the high expression of Talin-1 in ß-cells and that deficiency of Talin-1 reduces ß-cell proliferation, which leads to reduced ß-cell mass and insulin expression, thus causing glucose intolerance without affecting peripheral insulin sensitivity in mice. High-fat diet fed exerbates these phenotypes. Mechanistically, Talin-1 interacts with the E3 ligase smad ubiquitination regulatory factor 1 (Smurf1), which prohibits ubiquitination of the signal transducer and activator of transcription 3 (Stat3) mediated by Smurf1, and ablation of Talin-1 enhances Smurf1-mediated ubiquitination of Stat3, leading to decreased ß-cell proliferation and mass. Furthermore, haploinsufficiency of Talin-1 and Stat3 genes, but not that of either gene, in ß-cell in mice significantly impairs glucose tolerance and insulin expression, indicating that both factors indeed function in the same genetic pathway. Finally, inducible deletion Talin-1 in ß-cell causes glucose intolerance in adult mice. Collectively, our findings reveal that Talin-1 functions as a crucial regulator of ß-cell mass, and highlight its potential as a therapeutic target for DM patients.


Assuntos
Intolerância à Glucose , Talina , Adulto , Animais , Humanos , Camundongos , Proliferação de Células , Insulina/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Talina/genética , Talina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
J Mater Sci Mater Med ; 34(11): 54, 2023 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-37884819

RESUMO

A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs.


Assuntos
Adesões Focais , Nanoporos , Adesão Celular , Adesões Focais/metabolismo , Transdução de Sinais/fisiologia , Vinculina/metabolismo , Diferenciação Celular , Talina/metabolismo , Movimento Celular
13.
Cell Rep ; 42(11): 113321, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37874676

RESUMO

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.


Assuntos
Citoesqueleto , Adesões Focais , Adesões Focais/metabolismo , Ligação Proteica , Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Talina/metabolismo
14.
PLoS Comput Biol ; 19(10): e1011500, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37801464

RESUMO

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.


Assuntos
Actinas , Adesões Focais , Adesões Focais/metabolismo , Vinculina/metabolismo , Actinas/metabolismo , Integrinas/metabolismo , Matriz Extracelular/metabolismo , Adesão Celular/fisiologia , Talina
15.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 40(4): 645-653, 2023 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-37666754

RESUMO

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.


Assuntos
Simulação de Dinâmica Molecular , Talina
16.
Am J Respir Cell Mol Biol ; 69(6): 678-688, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37639326

RESUMO

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs in vitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI in vivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.


Assuntos
Lesão Pulmonar Aguda , Edema Pulmonar , Humanos , Lipopolissacarídeos/farmacologia , Calpaína/metabolismo , Talina/metabolismo , Pulmão/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Cadeias Leves de Miosina/metabolismo , Permeabilidade Capilar
17.
Cell Rep ; 42(8): 112936, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37552602

RESUMO

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.


Assuntos
Neoplasias da Mama , Fator de Crescimento Transformador beta , Humanos , Animais , Camundongos , Feminino , Fator de Crescimento Transformador beta/metabolismo , Neoplasias da Mama/patologia , Talina/metabolismo , Proteínas de Transporte , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Integrinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Linhagem Celular Tumoral , Metástase Neoplásica , Movimento Celular
18.
Cell Death Dis ; 14(7): 445, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-37460470

RESUMO

Breast cancer is the most common malignant cancer in women worldwide. Cancer metastasis is the major cause of cancer-related deaths. BCKDK is associated with various diseases, including proliferation, migration, and invasion in multiple types of human cancers. However, the relevance of BCKDK to the development and progression of breast cancers and its function is unclear. This study found that BCKDK was overexpressed in breast cancer, associated with poor prognosis, and implicated in tumor metastasis. The downregulation of BCKDK expression inhibited the migration of human breast cancer cells in vitro and diminished lung metastasis in vivo. BCKDK perturbed the cadherin-catenin complex at the adherens junctions (AJs) and assembled focal adhesions (FAs) onto the extracellular matrix, thereby promoting the directed migration of breast cancer cells. We observed that BCKDK acted as a conserved regulator of the ubiquitination of cytoskeletal protein talin1 and the activation of the FAK/MAPK pathway. Further studies revealed that BCKDK inhibited the binding of talin1 to E3 ubiquitin ligase-TRIM21, leading to the decreased ubiquitination/degradation of talin1. In conclusion, identifying BCKDK as a biomarker for breast cancer metastasis facilitated further research on diagnostic biomarkers. Elucidating the mechanism by which BCKDK exerted its biological effect could provide a new theoretical basis for developing new markers for breast cancer metastasis and contribute to developing new therapies for the clinical treatment of breast cancer patients.


Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Feminino , Humanos , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Adesões Focais/metabolismo , Neoplasias Pulmonares/secundário , Metástase Neoplásica/patologia , Talina
19.
Cell Mol Biol Lett ; 28(1): 56, 2023 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-37460977

RESUMO

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.


Assuntos
Melanoma , Talina , Humanos , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/genética , Integrinas/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Isoformas de Proteínas/metabolismo , Talina/genética , Talina/química , Talina/metabolismo , Linhagem Celular Tumoral/metabolismo
20.
Nat Commun ; 14(1): 4311, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37463895

RESUMO

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.


Assuntos
Actinas , Talina , Actinas/metabolismo , Talina/metabolismo , Vinculina/genética , Vinculina/metabolismo , Regulação Alostérica , Adesões Focais/metabolismo , Ligação Proteica
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